Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy is recommended for patients with muscle-invasive bladder cancer (MIBC). It has been shown that somatic deleterious mutations in ERCC2, gain-of-function mutations in ERBB2, and alterations in ATM, RB1, and FANCC are correlated with pathological response to NAC in MIBC. The objective of this study was to validate these genomic biomarkers in pretreatment transurethral resection material from an independent retrospective cohort of 165 patients with MIBC who subsequently underwent NAC and radical surgery. Patients with ypT0/Tis/Ta/T1N0 disease after surgery were defined as responders. Somatic deleterious mutations in ERCC2 were found in nine of 68 (13%) evaluable responders and two of 95 (2%) evaluable nonresponders (p = 0.009; FDR = 0.03). No correlation was observed between response and alterations in ERBB2 or in ATM, RB1, or FANCC alone or in combination. In an exploratory analysis, no additional genomic alterations discriminated between responders and nonresponders to NAC. No further associations were identified between the aforementioned biomarkers and pathological complete response (ypT0N0) after surgery. In conclusion, we observed a positive association between deleterious mutations in ERCC2 and pathological response to NAC, but not overall survival or recurrence-free survival. Other previously reported genomic biomarkers were not validated. Patient summary: It is currently unknown which patients will respond to chemotherapy before definitive surgery for bladder cancer. Previous studies described several gene mutations in bladder cancer that correlated with chemotherapy response. This study confirmed that patients with bladder cancer with a mutation in the ERCC2 gene often respond to chemotherapy.