Whole-exome sequencing of 109 micro-dissected pancreatic ductal adenocarcinoma (PDA) cases revealed significant genetic diversity and identified novel prognostic and therapeutic targets. Environmental stress and alterations in DNA repair genes were associated with distinct mutation spectra. Copy number alterations targeted multiple tumor suppressive/oncogenic loci, with MYC amplification uniquely linked to poor outcome and adenosquamous subtype. Novel mutated genes, such as RBM10 and KRAS, were identified, with RBM10 mutations associated with longer survival despite aggressive histological features. KRAS mutations were observed in over 90% of cases, with codon Q61 alleles selectively associated with improved survival. Oncogenic BRAF mutations were mutually exclusive with KRAS and defined sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signaling, chromatin remodeling, Hedgehog signaling, DNA repair, and cell cycle processes were observed. These findings provide insights into the genetic diversity of PDA and suggest potential therapeutic strategies.Whole-exome sequencing of 109 micro-dissected pancreatic ductal adenocarcinoma (PDA) cases revealed significant genetic diversity and identified novel prognostic and therapeutic targets. Environmental stress and alterations in DNA repair genes were associated with distinct mutation spectra. Copy number alterations targeted multiple tumor suppressive/oncogenic loci, with MYC amplification uniquely linked to poor outcome and adenosquamous subtype. Novel mutated genes, such as RBM10 and KRAS, were identified, with RBM10 mutations associated with longer survival despite aggressive histological features. KRAS mutations were observed in over 90% of cases, with codon Q61 alleles selectively associated with improved survival. Oncogenic BRAF mutations were mutually exclusive with KRAS and defined sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signaling, chromatin remodeling, Hedgehog signaling, DNA repair, and cell cycle processes were observed. These findings provide insights into the genetic diversity of PDA and suggest potential therapeutic strategies.