Molecular evolution describes how inherited DNA and/or RNA change over evolutionary time, and the consequences of this for proteins and other components of cells and organisms. Molecular evolution is the basis of phylogenetic approaches to describing the tree of life. Molecular evolution overlaps with population genetics, especially on shorter timescales. Topics in molecular evolution include the origins of new genes, the genetic nature of complex traits, the genetic basis of adaptation and speciation, the evolution of development, and patterns and processes underlying genomic changes during evolution.

The history of molecular evolution starts in the early 20th century with comparative biochemistry, and the use of "fingerprinting" methods such as immune assays, gel electrophoresis, and paper chromatography in the 1950s to explore homologous proteins. The advent of protein sequencing allowed molecular biologists to create phylogenies based on sequence comparison, and to use the differences between homologous sequences as a molecular clock to estimate the time since the most recent common ancestor. The surprisingly large amount of molecular divergence within and between species inspired the neutral theory of molecular evolution in the late 1960s. Neutral theory also provided a theoretical basis for the molecular clock, although this is not needed for the clock's validity. After the 1970s, nucleic acid sequencing allowed molecular evolution to reach beyond proteins to highly conserved ribosomal RNA sequences, the foundation of a reconceptualization of the early history of life. The Society for Molecular Biology and Evolution was founded in 1982. ^{[citation needed]}

Molecular phylogenetics uses DNA, RNA, or protein sequences to resolve questions in systematics, i.e. about their correct scientific classification from the point of view of evolutionary history. The result of a molecular phylogenetic analysis is expressed in a phylogenetic tree. Phylogenetic inference is conducted using data from DNA sequencing. This is aligned to identify which sites are homologous. A substitution model describes what patterns are expected to be common or rare. Sophisticated computational inference is then used to generate one or more plausible trees. ^{[citation needed]}

Some phylogenetic methods account for variation among sites and among tree branches. Different genes, e.g. hemoglobin vs. cytochrome c, generally evolve at different rates. These rates are relatively constant over time (e.g., hemoglobin does not evolve at the same rate as cytochrome c, but hemoglobins from humans, mice, etc. do have comparable rates of evolution), although rapid evolution along one branch can indicate increased directional selection on that branch. Purifying selection causes functionally important regions to evolve more slowly, and amino acid substitutions involving similar amino acids occurs more often than dissimilar substitutions.

Gene duplication can produce multiple homologous proteins (paralogs) within the same species. Phylogenetic analysis of proteins has revealed how proteins evolve and change their structure and function over time.

For example, ribonucleotide reductase (RNR) has evolved a multitude of structural and functional variants. Class I RNRs use a ferritin subunit and differ by the metal they use as cofactors. In class II RNRs, the thiyl radical is generated using an adenosylcobalamin cofactor and these enzymes do not require additional subunits (as opposed to class I which do). In class III RNRs, the thiyl radical is generated using S-adenosylmethionine bound to a [4Fe-4S] cluster. That is, within a single family of proteins numerous structural and functional mechanisms can evolve.

In a proof-of-concept study, Bhattacharya and colleagues converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. This demonstrates that only few mutations are needed to radically change the function of a protein. Directed evolution is the attempt to engineer proteins using methods inspired by molecular evolution.

Change at one locus begins with a new mutation, which might become fixed due to some combination of natural selection, genetic drift, and gene conversion. ^{[citation needed]}