Karyotypes of 16 Eupelminae species were analyzed. The haploid chromosome number was found to be much more diverse than the n = 5 previously recorded for three studied species. All species investigated of Eupelmus (Eupelmus), Eupelmus (Macroneura), Merostenus excavatus and three species of Anastatus have a karyotype of n = 5; n = 7 in Eupelmus (Episolindelia) linearis, n = 8 in Arachnophaga picardi, and n = 10 in Anastatus ruficaudus. The modal chromosome number is n = 5 and this is considered to be the plesiomorphic character state for the subfamily; karyotypes with higher chromosome numbers are hypothesized to be derived independently from the ancestral state by centric fission. This cytogenetical data provides important information on the subfamily systematics, especially the subgeneric classification of Eupelmus and generic placement of Eupelmus rostratus.