The available information on blood groups in the Chilean population is derived from studies on aboriginal cohorts and routine serological test results. The purpose of this study is to conduct a comprehensive analysis of genotypes, phenotypes and blood group alleles in donors from northern, central and southern Chile using molecular methods. Overall, 850 samples from donors in northern, central and southern Chile were genotyped. Allelic, genotypic and antigenic frequencies were calculated and compared among regions. Of these, 602 samples were analysed by haemagglutination, and discrepancies found between phenotypes and genotypes were investigated. The immunogenic potential of antigens was calculated by the Giblett equation, using the antigenic frequencies of donors from Santiago and the alloantibody frequencies of patients from the same region. Alleles of low prevalence, variant alleles and those responsible for the absence of high-prevalence antigens were found. Significant differences were observed between the antigenic frequencies of the three regions. Discrepancies between serologic and molecular results were mostly attributed to the molecular background affecting antigen expression. In the calculation of the immunogenic potential of antigens, the highest value was attributed to the Dia antigen. These findings represent the first molecular characterization of blood group antigens in Chileans. Our results highlight the necessity of using molecular tools to explore the genotypes underlying variant phenotypes, low-frequency antigens and antigens lacking specific antisera that cannot be detected by haemagglutination. Additionally, they emphasize the importance of understanding the distribution of blood groups among different populations.