BACKGROUND: Toll-like receptor 9 (TLR9) is a key immunotherapy target for glioblastoma (GBM). This study explored the correlation of TLR9 expression with tumor location and survival outcomes in patients with supratentorial GBM. METHODS: We retrospectively identified 46 patients with supratentorial GBMs and divided them into those with high TLR9 (TRL9(High)) and low TLR9 (TRL9(Low)) levels. The 2 groups were compared by patients' ages, sex, preoperative Karnofsky Performance Scale (KPS) score, resection extent, tumor location, progression-free survival (PFS), and overall survival (OS). RESULTS: The TLR9 expression percentages for the GBM specimens were TRL9(High): 72% (33/46) and TRL9(Low): 28% (13/46). The 2 groups showed no differences in patient age (P = 0.147) and sex (chi(2) = 0.002, P = 0.966), preoperative KPS score (chi(2) = 0.033, P = 0.855), or resection extent (chi(2) = 2.405, P = 0.121). Location differed significantly, with 85% (11/13) of TRL9(Low) tumors in the left hemisphere and 45% (15/33) of TRL9(High) tumors on the same side (chi(2) = 5.82, P = 0.016). The TRL9(Low) group had a median PFS of 612 days (range, 77-926 days), significantly longer than the TRL9(High) group (355 days; range, 105-861 days; P = 0.042). Median OS of the TRL9(Low) group (733 days; range, 163-969 days) was also significantly longer than in the TRL9(High) group (396 days; range, 135-1024 days; P = 0.020). CONCLUSIONS: Patients with TRL9(Low) supratentorial GBM tend to have longer survival than those with higher TLR9 expression. Such tumors show a location preference for the left hemisphere.