BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignant form of cancer with the worst survival rate and an extremely low rate of response to treatments. The development and molecular characterization of pancreatic cancer cell lines (PCCLs) are essential for studying the biology of highly aggressive pancreatic adenocarcinoma. METHODS: We applied whole exome sequencing (WES) and RNA-seq to identify molecular characteristics of 26 newly established PCCLs. Eighteen clinically relevant anti-cancer drugs were used to assess highly heterogeneous drug responses across the 26 cell lines. RESULTS: We confirmed that common driver mutations of PDAC were well retained in our cell lines through WES analysis. Transcriptomic analysis identified two representative clusters that correlated with responses to certain drugs. By using Moffitt's classification method, the two clusters, C1 and C2, showed comparable expression patterns to "Basal-like" and "Classical" types, respectively. Drug screening results showed varying responses among different cell lines. In our cohort, C2 displayed greater sensitivity to anti-cancer drugs compared to C1. Furthermore, drugs targeting similar molecular pathways exhibited corresponding reactions among cell lines. CONCLUSIONS: Our results underscored that transcriptomic features of pancreatic cancer correlate with drug sensitivity rather than with the effects of targeted drugs. Cell lines are useful in vitro model systems for studying the molecular mechanisms of PDAC.