In previous studies, major histocompatibility complex (MHC) class II DP, DQ, and DR families of genes were characterized in different primate species mostly on the basis of their second exon sequences. Resemblances were found between Old World monkey (OWM) and New World monkey (NWM) genes and were interpreted as being the result of transspecies evolution. Subsequent analysis of intron sequences of catarrhine and platyrrhine DRB genes, however, revealed that the amplifiable genes were not, in fact, orthologous. To test other DRB genes and other families of the class II region Southern blot hybridizations were carried out with tamarin genomic DNA using probes specific for the third exons of the tamarin DQA, DQB, DPB, and DRB genes. The hybridizing bands were extracted from the gel and the third exons of the genes were amplified by PCR, cloned, and sequenced. With two exceptions, all NWM class II genes were found to group separately from the human sequences. Only the sequences of one nonfunctional DQB locus appeared to be more closely related to human genes than to other platyrrhine DQB genes. In the DRB family one gene was found that grouped with sheep and strepsirhine DRB sequences and might represent an old gene lineage. To extend the sequences to the second exon, long PCRs were performed on tamarin genomic DNA. This approach was successful for five of the ten third exon sequences. From these data, we conclude that at least the functional MHC class II genes have expanded independently in catarrhines and platyrrhines.