Infection with Blastocystis sp. has been reported in free-living and captive non-human primates (NHPs); however, surveys on Blastocystis sp. from north-western South America are scarce. This study aimed to identify Blastocystis sp. in free-ranging NHPs living in Colombia. A total of 212 faecal samples were collected from Ateles hybridus, Cebus versicolor, Alouatta seniculus, Aotus griseimembra, Sapajus apella, and Saimiri cassiquiarensis. Smears and flotation were used for morphological identification. For samples microscopically classified as positive for Blastocystis sp., we used conventional PCR to amplify and sequence two regions of the SSU rRNA gene and used Maximum Likelihood methods and Median Joining Network analyses for phylogenetic analyses. Via microscopy, 64 samples were Blastocystis sp. positive. Through molecular analyses, 18 sequences of Blastocystis sp. subtype 8 (ST8) were obtained. Strain and allele assignment together with a comparative phylogenetic approach confirmed that the sequences were ST8. Alleles 21, 156, and 157 were detected. Median Joining network analyses showed one highly frequent haplotype shared by specimens from Colombia and Peru and close relationships between haplotypes circulating in NHPs from Colombia, Ecuador, Brazil, and Mexico. This survey could support the elaboration of a more accurate epidemiological picture of the Blastocystis sp. infecting NHPs.