Background: Nucleotide mutations in theABOgene may reduce the activity of glycosyltransferase, resulting in lower levels of A or B antigen expression in red blood cells. Six known splice sites have been identified according to the database of red cell immunogenetics and the blood group terminology of the International Society of Blood Transfusion. Here, we describe six distinct splice site variants in individuals with ABO subtypes.Methods: The ABO phenotype was examined using a conventional serological method. A polymerase chain reaction sequence-based typing method was used to examine the whole coding sequence of theABOgene. TheABOgene haplotypes were studied using allele-specific primer amplification or cloning technology. In silico analytic tools were used to assess the functional effect of splice site variations.Results: Six distinct variants in theABOgene splice sites were identified in nine individuals with ABO subtypes, including c.28 + 1_2delGT, c.28 + 5G > A, c.28 + 5G > C, c.155 + 5G > A, c.204-1G > A and c.374 + 5G > A. c.28 + 1_2delGT was detected in an A_windividual, while c.28 + 5G > A, c.28 + 5G > C, and c.204-1G > A were detected in B_elindividuals. c.155 + 5G > A was detected in one B₃and two AB₃individuals, whereas c.374 + 5G > A was identified in two A_elindividuals. Three novel splice site variants (c.28 + 1_2delGT, c.28 + 5G > A and c.28 + 5G > C) in theABOgene were discovered, all of which resulted in low antigen expression. In silico analysis revealed that all variants had the potential to alter splice transcripts.Conclusions: Three novel splice site variations in theABOgene were identified in Chinese individuals, resulting in decreased A or B antigen expression and the formation of ABO subtypes.