The tripartite motif 5 protein (TRIM5) has been extensively studied in view of its ability to restrict retroviruses in mammalian hosts. The B30.2 domain, encoded by exon 8 of TRIM5, contains the major restriction determinants. We have analyzed the genetic diversity of the TRIM5 B30.2 domain in a wide range of New World primates (NWP). The TRIM5 region encoding the B30.2 domain of 35 animals, representing all NWP families and 10 genera, was PCR-amplified, sequenced and analyzed at the amino acid level. Comparisons were carried out with available GenBank data; analyses were carried out with a dataset of 44 representative sequences of 32 NWP species and 15 genera, with a human B30.2 sequence as outgroup. A high genetic diversity was observed, both with respect to length and amino acid substitutions, mainly at the three variable regions of this domain associated with the restriction phenotype. Phylogenetic reconstructions based on B30.2 DNA differed from the consensus NWP topology due to positive selection along different lineages and definite codon positions, with robust evidence either with a complete or a pruned dataset. This was especially evident in codons 406 and 496, consistently demonstrated with all methods. Positive selection was virtually absent in all NWP species when analyzing intra-specific polymorphisms except for Saguinus labiatus. Our findings indicated that NWP TRIM5 proteins have been subjected to selection, probably by retroviruses and/or retroelements. We anticipate that the diversity of NWP TRIM5 is indicative of disparate retroviral restriction phenotypes representing a plentiful source of factors countering HIV infection.