Axial spondyloarthritis (axSpA) is a chronic, immune-mediated disease characterized by inflammatory axial skeleton involvement and extra-musculoskeletal manifestations. The continuum of axSpA ranges from nonradiographic axSpA (nr-axSpA) to ankylosing spondylitis, also known as radiographic axSpA; the latter is defined by definitive radiographic sacroiliitis. Human leukocyte antigen B27 (HLA-B27) is a genetic marker strongly associated with axSpA; it aids in the diagnosis of axSpA, and its absence leads to delay in diagnosis. For HLA-B27-negative patients, disease pathogenesis is poorly understood, signs and symptoms are frequently underrecognized, and diagnosis and treatment are commonly delayed. The proportion of HLA-B27-negative patients may be higher among non-White patients and those with nr-axSpA, who can face additional diagnostic challenges related to lack of definitive radiographic sacroiliitis. In this narrative review, we discuss the role of HLA-B27 in the diagnosis and pathogenesis of axSpA and highlight various pathways and genes that may be related to axSpA pathogenesis in HLA-B27-negative patients. We also emphasize the need to characterize gut microbial communities in these patients. Adequate understanding of clinical and pathological features underlying HLA-B27-negative patients with axSpA will improve diagnosis, treatment, and outcomes for this complex inflammatory disease.