The genomic heterogeneity of pancreatic ductal adenocarcinoma (PDAC) is becoming increasingly appreciated. We aimed to evaluate the ability of a triple biomarker panel (S100A4, Ca-125, and mesothelin) to predict: (i) genetic PDAC subtypes; (ii) clinical phenotypes; and (iii) the optimal treatment strategy (neoadjuvant vs. surgery-first) in resectable and borderline resectable PDAC. Patients who underwent resection for resectable and borderline resectable PDAC were included from one single-institutional cohort and one multi-institutional cohort from the Australian Pancreatic Genome Initiative (APGI). Tumors were immunohistochemically evaluated for S100A4, Ca-125, and mesothelin, and a subset from the APGI cohort underwent RNA sequencing. This study included 252 and 226 patients from the single institution and the APGI cohorts, respectively. Triple-negative biomarker status correlated with non-squamous PDAC genotypes (p = 0.020), lower rates of distant recurrence (p = 0.002), and longer median overall survival (mOS) with the surgery-first approach compared with neoadjuvant treatment (33.3 vs. 22.2 mths, p = 0.038) in resectable PDAC. In contrast, the triple-positive disease was associated with longer mOS with neoadjuvant treatment compared with the surgery-first approach (29.5 vs. 13.7 mths, p = 0.021) in resectable and borderline resectable PDAC. In conclusion, the triple biomarker panel predicts genetic PDAC subtypes, clinical phenotypes, and optimal treatment strategies in resectable and borderline resectable PDAC.