The Indian blood group system (ISBT 023) comprises one low-prevalence antigen, In^a (IN:1), and five high-prevalence antigens: In^b (IN:2), INFI (IN:3), INJA (IN:4), INRA (IN:5), and INSL (IN:6). The antigens are located on the single-pass trans-membrane glycoprotein encoded by the CD44 gene. The present study was designed to identify the prevalence of the INRA– (IN:−5) phenotype and the frequency of its associated allele (IN*02.– 05) to inform us of the probability of finding antigen-negative donors and to assess the risk of antibody formation in transfusion recipients. Buffy coats were extracted from EDTA-anticoagulated whole blood samples, collected with consent from 5261 random blood donors in Surat, Gujarat, India. Standard serologic methods were performed with a modification allowing the use of antiserum generated by recycling the antibody augmented from the test already performed. A real-time polymerase chain reaction– based assay was devised to genotype c.449G>A (p.Arg150His) single nucleotide variation in exon 5 of the CD44 gene. None of the 411 donors tested by serology or the 5261 donors tested molecularly were positive for the IN:−5 phenotype or the allele (IN*02.–05), respectively. The allele frequency estimate ranged from less than 1 in 10,522 (0.01%) to 1 in 3203 alleles (0.03%) in the study cohort (95% confidence interval, Poisson distribution). The absence of this rare allele in the present survey could be due to an ethnic difference, since the donors mostly came from the Hindu community, and the only case of the IN:−5 phenotype was found in the Muslim community. The p.150His variant may be either restricted to the index case family or only found in the Muslim community. Further studies in local subpopulations may provide more information on the frequency of p.150His and its immunogenicity in transfusion recipients if occurring among blood donors.