Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal malignancies in the world, often diagnosed at an advanced stage, resistant to conventional chemotherapy and having high invasive and metastatic potential. The mechanism of drug resistance of PDA is still not clear. In the present study, we established two novel pancreatic cancer cell lines PAXC-002 and PAXC-003 from human primary xenograft models. The cell lines were characterized by morphology, karyotype, pancreatic cancer marker and short tandem repeat (STR) analysis, and growth kinetics and tumorigenicity. The in vitro anti-proliferation test revealed that PAXC-002 cell was intrinsically resistant to the standard of care chemotherapy-gemcitabine, compared with that of PAXC-003 and other widely used pancreatic cancer cell lines. Interestingly, the gemcitabine resistant PAXC-002 cell line was more potent in forming colonies in 3-Dimensional matrigel culture conditions and had a higher percentage of CD133 positive cells, which is recognized as a cancer stem cell marker, compared to the gemcitabine-sensitive PAXC-003 cell line. In this study, we present two novel pancreatic cancer cell lines which could be used for gemcitabine resistance investigation, mechanism identification of pancreatic cancer and anticancer drug screening. The preliminary data indicate that the drug resistance of pancreatic carcinoma cells is associated with a cancer stem cell-like phenotype.