The presence of several Mycobacterium species was determined in 68 New World monkeys kept captive in the Cali Zoo. One hundred and thirty-three gastric lavage and blood samples were evaluated for mycobacterial presence by Ziehl-Neelsen (ZN) staining, culture and PCR amplification of the Mycobacterium tuberculosis Mtp40 species-specific gene. Mycobacteria other than tuberculosis (MOTT) were identified by PCR restriction fragment length polymorphism (RFLP). Different species of mycobacteria were detected in 65% of the primate population studied by Alpha Antigen PCR. Eleven percent were positive for Mtp40 PCR amplification, being diagnosed as having M. tuberculosis, and acid-fast bacilli were observed in 23% by ZN staining. MOTT were isolated from samples taken from 37 primates by culturing; according to the RFLP analysis, three strains were classified as belonging to the MAISS complex (Mycobacterium avium-intracellulare-scrofulaceum-simiae) and eight more, isolated from soil inside the cages, were categorized as environmental contaminants. Mycobacterium spp. were detected in 13 different New World primate species showing that PCR amplification of the Mtp40 gene is a better tool than culture for M. tuberculosis detection in captive animals and that RFLP is a useful technique for MOTT identification.