This study examined the distribution and disease associations of non-HLA-B27 HLA-B alleles in Romanian spondyloarthritis (SpA) patients, aiming to address the underrepresentation of Eastern European populations in immunogenetic research. Methods: We analyzed 263 HLA-B27-negative patients from Northeastern Romania fulfilling ASAS criteria. HLA-B genotyping was performed at two-digit resolution, and allele distributions were compared with two Romanian HLA-B27-negative control groups ( = 335 and = 1705 cases), using chi-square testing and logistic regression. Compared to controls, HLA-B47 ( = 0.0007) and HLA-B54 ( = 0.0013) were significantly enriched, while HLA-B40 was underrepresented ( = 0.0287). Notably, HLA-B54 was observed exclusively in axial SpA. Within the cohort, both HLA-B13 and HLA-B57 alleles were associated with psoriasis, while HLA-B37 and HLA-B41 alleles were clustered within the reactive arthritis group. The HLA-B35 and HLA-B18 alleles were the most frequently observed alleles across most clinical phenotypes. When comparing the frequency of HLA-B associations, the most common genotypes among SpA patients were B08-B18, B13-B35, and B35-B51. Notably, B08-B18 was more frequent in patients with radiographic sacroiliitis grade ≥ 2, while B35-B51 was more frequent in those with confirmed systemic inflammation, as indicated by elevated CRP or ESR levels. Analysis of peptide-binding patterns revealed a cluster of risk alleles, HLA-B08, B18, B35, B40, and B54, sharing similar features, distinct from the canonical profile of B27. These findings highlight the contribution of non-B27 HLA-B alleles to SpA susceptibility in an Eastern European population and support the notion that HLA-B27-negative SpA may represent a distinct clinical and immunological entity, driven by alternative pathogenic mechanisms. They also emphasize the importance of population-specific immunogenetic profiling and support expanding genetic characterization in HLA-B27-negative patients.