Purpose <p>To explore the value of laboratory serum markers and magnetic resonance imaging(MRI) features in the preoperative differential diagnosis of pancreatic ductal adenocarcinoma (PDAC) in periampullary carcinoma (PAC).</p> Methods <p>A retrospective analysis of clinical data from 105 PAC patients who underwent pancreaticoduodenectomy was conducted, including 33 cases of PDAC (observation group) and 72 cases of non-PDAC (control group), with 25 cases of ampullary carcinoma, 38 cases of distal bile duct carcinoma, and 9 cases of periampullary duodenal carcinoma. Laboratory serum markers, MRI features, and pathological diagnosis data were compared between the two groups to analyze the value of laboratory serum markers and MRI features for differential diagnosis of PDAC within PAC.</p> Results <p>Compared to the control group, the observation group had higher proportions in total bilirubin, direct bilirubin, carcinoembryonic antigen(CEA), carbohydrate antigen 19 − 9 (CA19-9), quadruple duct sign, pancreatobiliary junction angle, main pancreatic duct diameter, pancreatic head side branch duct dilation, and hypovascular mass in pancreatic head, consistency of imaging and pathology diagnosis, perineural invasion (<i>p</i> &lt; 0.05).The common bile duct diameter was smaller in the observation group (<i>p</i> &lt; 0.05). The sensitivities of CA19-9, main pancreatic duct diameter, pancreatic head side branch duct dilation, and hypovascular mass in pancreatic head in PDAC within PAC are 53.8%, 51%, 84.6%, and 81.1%, respectively; the specificities are 81.8%, 87%, 86.1%, and 95.6%, respectively; and the areas under the ROC curve are 0.74, 0.749, 0.806, and 0.906, respectively.</p> Conclusion <p>In the preoperative diagnosis of PAC, CA19-9, main pancreatic duct diameter, hypovascular mass in pancreatic head, and pancreatic head side branch duct dilation are effective indicators for distinguishing PDAC.</p>