Background and Objective <p>Adjuvant nivolumab has demonstrated clinical benefit in urothelial carcinoma; however, emerging evidence suggests that its efficacy may differ between bladder cancer and upper tract urothelial carcinoma (UTUC), potentially reflecting differences in genomic profiles and the tumor microenvironment (TME). This study aimed to evaluate the association between key oncogenic mutations and the TME characteristics in UTUC, with a particular focus on the impact of neoadjuvant chemotherapy (NAC).</p> Methods <p>We analyzed tumor specimens from 32 patients who underwent nephroureterectomy for UTUC. Patients were categorized into NAC and non-NAC groups. Oncogenic mutations (<i>TP53</i>, <i>FGFR3</i>, and <i>RAS</i>) were assessed in all patients, and immune cell densities (CD3⁺, CD4⁺, CD8⁺, Foxp3⁺, and CD204⁺) in intratumoral and peritumoral regions were quantified using multiplex fluorescent immunohistochemistry.</p> Results <p><i>TP53</i>, <i>FGFR3</i>, and <i>RAS</i> mutations were identified in 29%, 31%, and 34% of patients, respectively. NAC was administered to 22% of patients. The NAC group demonstrated significantly higher densities of intratumoral CD4⁺ T cells (<i>p</i> = 0.0201) and a trend toward increased CD8⁺ T cells (<i>p</i> = 0.0614). Among patients with <i>TP53</i> mutations, NAC was associated with significantly higher intratumoral CD4⁺ T cell density (<i>p</i> = 0.0369) compared without NAC. In contrast, <i>FGFR3</i>-mutant tumors exhibited consistently low immune cell infiltration, irrespective of NAC status.</p> Conclusions <p><i>TP53</i>-mutant tumors from patients who received NAC were associated with higher intratumoral CD4⁺ T cell density, whereas <i>FGFR3</i>-mutant tumors exhibited persistently low immune-cell infiltration irrespective of NAC exposure. These findings highlighted distinct mutation-associated TME features in UTUC.</p>