Third-generation sequencing (also known as long-read sequencing) is a class of DNA sequencing methods that have the capability to produce substantially longer reads (ranging from 10 kb to >1 Mb in length) than second generation sequencing, also known as next-generation sequencing methods. These methods emerged in 2008, characterized by technologies such as nanopore sequencing or single-molecule real-time sequencing, and continue to be developed. The ability to sequence longer reads has critical implications for both genome science and the study of biology in general. In structural variant calling, third generation sequencing has been found to outperform existing methods, even at a low depth of sequencing coverage. However, third generation sequencing data have much higher error rates than previous technologies, which can complicate downstream genome assembly and analysis of the resulting data. These technologies are undergoing active development and it is expected that there will be improvements to the high error rates.

Sequencing technologies with a different approach than second-generation platforms were first described as "third-generation" in 2008–2009.

There are several companies currently at the heart of third generation sequencing technology development, namely, Pacific Biosciences, Oxford Nanopore Technology, Quantapore (CA-USA), and Stratos (WA-USA). These companies are taking fundamentally different approaches to sequencing single DNA molecules.

PacBio developed the sequencing platform of single molecule real time sequencing (SMRT), based on the properties of zero-mode waveguides. Signals are in the form of fluorescent light emission from each nucleotide incorporated by a DNA polymerase bound to the bottom of the zL well.

Oxford Nanopore's technology involves passing a DNA molecule through a nanoscale pore structure and then measuring changes in electrical field surrounding the pore; while Quantapore has a different proprietary nanopore approach. Stratos Genomics spaces out the DNA bases with polymeric inserts, "Xpandomers", to circumvent the signal to noise challenge of nanopore ssDNA reading.

Also notable is Helicos's single molecule fluorescence approach, but the company entered bankruptcy in the fall of 2015.

In comparison to the second generation of sequencing technologies, third generation sequencing has the obvious advantage of producing much longer reads. It is expected that these longer read lengths will alleviate numerous computational challenges surrounding genome assembly, transcript reconstruction, and metagenomics among other important areas of modern biology and medicine.

It is well known that eukaryotic genomes including primates and humans are complex and have large numbers of long repeated regions. Short reads from second generation sequencing must resort to approximative strategies in order to infer sequences over long ranges for assembly and genetic variant calling. Pair end reads have been leveraged by second generation sequencing to combat these limitations. However, exact fragment lengths of pair ends are often unknown and must also be approximated as well. By making long reads lengths possible, third generation sequencing technologies have clear advantages.