An association between human leukocyte antigen-DRβ1*04 and rheumatoid arthritis (RA) has been known for more than 25 years. It has been observed in many different populations, and it accounts for approximately one-third of the genetic component of RA susceptibility. Our aim was to study the distribution of HLA-DRβ1 alleles in well-characterized RA patients from Western India. Polymerase chain reaction-based sequence-specific oligonucleotide probing (PCR-SSOP) technique was used to identify HLA-DRβ1 alleles among 80 clinically well-defined patients and 90 normal controls from same ethnicity. A significant increase in the frequency of DRβ1*04 was observed among RA patients (PF% 30 vs. 7.7, OR 4.959, p value 0.00018), whereas DRβ1*03 and *14 were significantly decreased among patients when compared with controls (DRβ1*03, PF% 8.75 vs. 26.6, OR 0.2637, p value 0.00253; DRβ1*14, PF% 17.5 vs. 30.0, OR 0.4949, p value 0.05722). Our results suggest that DRβ1*04 was strongly associated with well-characterized RA patients from Western India, whereas DRβ1*03 and *14 may be protective alleles for RA. The identification of susceptible allele in patients with RA may help physician to make early decisions regarding initiation of early intensive therapy with disease modifying anti-rheumatic drugs and biological agents to decrease disability in RA patients.