Blastocystis spp. has become one of the protozoans arousing the greatest scientific interest because of the controversy surrounding its biology; it is currently considered one of the most prevalent organisms in humans and animals worldwide. Such prevalence increases, especially in tropical countries where infection rates are high, highlighting the need to conduct studies focused on understanding this protozoan's biology. Interestingly, molecular tools are emerging as the best option for diagnosing this infection. This study was thus aimed at conventional PCR molecular detection and characterisation of Blastocystis spp. in human faecal samples from Ibagué, Colombia, using primers targeting the small subunit ribosomal ribonucleic acid (rRNA) gene. One hundred human faecal samples with confirmed Blastocystis spp. were studied, revealing the following subtype genetic diversity: ST1 50%, ST2 33% and ST3 17%. The results contributed to the limited information available regarding Blastocystis spp. in Colombia and created a reference point for further studies in the region.