Abstract Background The HLA-B*57:01 allele is associated with a hypersensitivity reaction to abacavir. Due to the lack of knowledge of HLA-B*57:01 prevalence in Colombia, routine screening is not performed and is not recommended by the national guidelines. We aimed to determine the prevalence of HLA-B*57:01 in HIV population from Colombia. Methods This cross-sectional study included naïve HIV-infected adults from 13 cities of the country. The presence of HLA-B*57:01 was determined by using SSP-PCR in blood samples. Prevalence rates were stratified by sex, race, and region of origin. Results HLA-B*57:01 allele prevalence in Colombian HIV-infected individuals was 2.7%. When stratifying for the race, the prevalence was 4% for whites, 2.6% for other race (mainly mestizo), and 1.9% for Afro-Colombians. The prevalence varied from 0% up to 11.4% depending on the department of origin. The highest prevalence rates were found in Caldas (11.4%), Antioquia (5%), Risaralda (4.8%), and Valle del Cauca (4.3%). When distributed by country zones, the central, with a racial predominance of Caucasians and mestizos, was the highest (6.0%, 0R = 4.1, CI 1.2–12.8, p = 0,016). Conclusions The overall prevalence of HLA-B*57:01 in Colombia was lower than the reported rates for other Latin American countries such as Brazil, Costa Rica, and Argentina, but similar in comparison to Chile and Mexico. The diversity in the racial and ethnic heritage shown in our data supports the recommendation to implement routine screening for the HLA-B*57:01 allele before initiation of abacavir-containing antiretroviral therapy in the Colombian HIV management guidelines.