Neoadjuvant chemotherapy (NAC) is often the treatment of choice for borderline resectable and locally advanced invasive pancreatic ductal adenocarcinoma (PDAC); however, most cancers only partially respond to therapy. We hypothesized that the location of residual neoplastic cells in resected specimens following NAC could provide a clue as to the mechanisms of resistance. PDAC cells invade the stroma but can also invade back into and spread via the pancreatic ducts, which has been referred to as "cancerization of ducts" (COD). We compared the responsiveness to chemotherapy between PDAC cells in the stroma and PDAC cells in the duct. Pancreatic resections from a total of 174 PDAC patients (NAC, n = 97; immediate surgery, n = 77) were reviewed. On hematoxylin and eosin sections, COD was identified at the same prevalence in both groups (NAC: 50/97 cases, 52%; immediate surgery: 39/77 cases, 51%; p = 0.879, Fisher's exact test). However, using quantitative image analysis of CK19 immunohistochemistry, we found that the proportion of cancer cells that were intraductal was significantly different between the NAC and immediate surgery groups (median; 12.7% vs. 1.99%, p < 0.0001, Mann-Whitney U test). This proportion was highest in patients with marked therapy responses (36.2%) compared with patients with moderate or poor responses (7.21 & 7.91%). In summary, our data suggest that intraductal components in PDAC are less responsive to chemotherapy than the remainder of the tumor, which could have important implications for therapeutic resistance.