On a global scale, potato (Solanum tuberosum L.) plays an important role in tackling the threat of food insecurity due to its high yield and broad global acceptance. However, pathogens threaten potato production, causing direct yield loss and rendering potatoes tubers unmarketable. Columbia root-knot nematode (CRKN, Meloidogyne chitwoodi) parasitizes potato plants and causes small brown dots in the tuber flesh that dramatically reduce the market value of the crop. In the Pacific Northwest (PNW) two races and a pathotype of M. chitwoodi exist: Race 1 and Race 2 and Race 1Roza. The races of M. chitwoodi are primarily identified based on a differential host test. To understand the phylogeny of M. chitwoodi and to develop molecular markers to differentiate different races, we sequenced the genomes of M. chitwoodi Race 1, Race 2 and Race 1Roza using Illumina and PacBio sequencing. Each genome was assembled and annotated. Comparisons of syntenies and orthologs elucidate the complex evolutionary history of this species and facilitate molecular marker development and analysis of host plant resistance to these root-knot nematodes. Based on the genome comparisons we successfully developed molecular markers that can differentiate M. chitwoodi races from DNA extracted from nematode eggs as well as soil samples. The newly developed markers have potential in disease diagnostics