| Summary: | Background: The present study aims to perform an epidemiological and molecular characterization of <i>Blastocystis</i> infection in a child population attending daycare centers of Medellín, Colombia. Methods: A total of 265 children aged 0–5 years were enrolled in five children’s centers in urban sectors of Medellín, northwestern Colombia. Stool samples were taken to identify intestinal parasites by direct examination, Ritchie–Frick concentration, and molecular identification of <i>Blastocystis</i> by conventional PCR and subtype (ST) identification by PCR barcoding with subsequent phylogenetic reconstruction. Kappa index was calculated to evaluate the agreement between microscopy and PCR for the diagnosis of <i>Blastocystis</i>. Results: The prevalence of intestinal protozoa was 36.6% (97/265), with <i>Blastocystis</i> as the most frequent parasitic protozoan at 15.8% (42/265), followed by <i>Giardia intestinalis</i> at 15.5% (41/265) and <i>Endolimax nana</i> at 15.1% (40/265). The prevalence of <i>Blastocystis</i> by PCR was 53.2% (141/265), the subtypes identified were ST3 at 30.5% (18/59), ST2 at 23.7% (14/59), ST1 at 20.3% (12/59), and with less frequency, ST4 at 5.1% (3/59), ST6 at 1.7% (1/59) and ST16 at 15.3% (9/59) allele 162. Conclusion: This study provides the first genetic characterization of <i>Blastocystis</i> subtypes circulating in a population of Medellín, Colombia, and also updates the epidemiology of <i>Blastocystis</i> subtypes in the world with the first identification of ST16 in humans.
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