In total, 140 populations of the genus Meloidogyne collected from representative horticultural regions of Spain and maintained at the Department of Agroecology, Institute of Agrarian Sciences, Madrid were characterized using perineal morphology, sequenced characterized amplified region polymerase chain reaction with species-specific primers, and bioassay. Using a modified North Carolina differential host test as a basis, 13 Meloidogyne populations did not fit into the published race scheme. These populations had very limited host ranges, reproducing only on susceptible tomato cv. Marmande and sometimes also on tobacco cv. NC95 and the pepper cv. Sonar in the case of Meloidogyne arenaria. They did not reproduce on cotton or peanut. The species and new races were identified as M. incognita races 5 (6 populations) and 6 (2 populations) and M. javanica race 5 (2 populations), with new records of M. arenaria race 3 (2 populations) and M. javanica race 1 (23 populations) for Spain.