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Root-knot nematodes (Meloidogyne spp.) are the most common major pathogens of many crops throughout the world, impacting both the quantity and quality of marketable yields. In this study, a total of 244 root-knot nematode populations from various hosts from 39 counties in Arkansas were tested to determine the species diversity. Molecular characterization was performed on these populations by DNA sequencing of the ribosomal DNA 18S-ITS-5.8S, 28S D2/D3 and a mitochondrial DNA fragment flanking cytochrome oxidase gene subunit II - the intergenic spacer. Five species were identified, including M. incognita (Kofoid & White, 1919) Chitwood, 1949 from soybean, cotton, corn and various vegetables (232 samples); M. hapla Chitwood, 1949 from rose (1 sample); M. haplanaria Eisenback, Bernard, Starr, Lee & Tomaszewski, 2003 from okra, tomato, peanut, Indian hawthorn, ash, willow and elm trees (7 samples); M. marylandi Jepson & Golden in Jepson, 1987 from grasses (3 samples); and M. partityla Kleynhans, 1986 from pecan (1 sample) through a combined analysis of DNA sequencing and PCR by species-specific primers. Meloidogyne incognita is the most abundant species that was identified in 95% samples and was the only species in field crops including soybean and cotton, except for one population of M. haplanaria from soybean in Logan County (TK201). Species-specific primers were used to verify M. incognita through PCR by species-specific primers. Unlike historical data, M. arenaria, M. javanica and M. graminis were not detected from any of the samples collected during this study. This result is essential for effective and sustainable management strategies against root-knot nematodes in Arkansas. [ABSTRACT FROM AUTHOR]