BACKGROUND: There is increased use of neoadjuvant fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) in the management of localized pancreatic ductal adenocarcinoma (PDAC), yet there are few validated biomarkers of treatment response. STUDY DESIGN: Consecutive patients (n = 196) with resectable, borderline resectable or locally advanced PDAC (2012-2019) receiving FOLFIRINOX as initial treatment and with targeted sequencing of a pretreatment biopsy were identified in a prospective institutional database. Genomic alterations were determined in the 4 driver mutations (KRAS, TP53, CDKN2A, SMAD4), and associations between genomic alterations and clinical outcomes were assessed. RESULTS: Alterations in KRAS (n = 172, 87.8%) and TP53 (n = 131, 66.8%) were common; alterations in CDKN2A (n = 49, 25.0%) and SMAD4 (n = 36, 18.4%) were less frequently observed. A total of 105 patients (53.6%) were able to undergo resection, of whom 8 (7.6%) had a complete/near-complete pathologic response. There were no somatic alterations associated with major pathologic response. Alterations in SMAD4 were associated with a lower rate of surgical resection (27.8% vs 59.4%, p < 0.001); this was additionally observed in a multivariable regression model accounting for resectability status (OR 0.35, 95% confidence interval [CI] 0.15-0.85). Thirty-three patients (16.8%) developed metastatic disease while on neoadjuvant therapy. SMAD4 alterations were associated with a significant risk of metastatic progression on therapy when controlling for resectability status (OR 3.31, 95% CI 1.44-7.60). CONCLUSIONS: SMAD4 alterations are associated with more frequent development of metastasis during neoadjuvant FOLFIRINOX and lower probability of reaching surgical resection. Evaluation of alternative chemotherapy regimens in patients with SMAD4 alterations will be important to distinguish whether this represents a prognostic or predictive biomarker.