Background: Several factors are involved in pancreatic cancer’s (PC) resistance to therapy. Cancer Stem cells (CSCs) represent a small and unique population of cancer cells, that are resistant to chemotherapy and radiotherapy and have the capability to differentiate and produce further resistant clones. Our hypothesis was that stem cell frequency would differ in treated vs. untreated pancreatic cancers. Methods: Resected FFPE PC specimens were collected under IRB- approved protocols and were separated into two group categories: cases that received neoadjuvant therapy (NA) and matched control cases with no treatment prior to surgical resection (SU). Immunostaining with CD24 and ALDH1 was performed using the DAKO FLEX SYSTEM. Each slide was scanned using the Aperio Whole Digital Imaging System. The digital scans were analyzed using the standard positive pixel algorithm for immunostains (brown positive, blue negative) that gives a percent of positive pixels in the cross section of the tissue. Care was taken to exclude false-positive staining sections of intact physiologic acinar and islet cells. Positivity data was compared between the two groups using a two-sample t-test using unequal or equal variances where an F-test deemed each appropriate. Results: Twenty cases were examined (10 NA and 10 SU). Patients in the NA arm received the following treatments (4 FOLFIRINOX, 2 nab-Paclitaxel + gemcitabine, 2 treated with concurrent gemcitabine/radiotherapy and 2 patients’ details are not available). Using two-sample t-test analysis, we found a statistically significant difference of median expression (percent positive pixels) of CD24 (20.5% vs. 66.9% expression, P <0.01,) and ALDH1 (41.5% vs. 60.5%, P= 0.04) between NA vs. SU respectively. This small study demonstrates lower expression of CSCs markers in patients receiving NA therapy in comparison to SU treated patients, indicating an effect from neoadjuvant therapy on CSCs. Conclusions: This is the first analysis of CSCs markers in patients with PC receiving NA therapy compared to surgery. In this small group, CSCs markers reflecting CSCs numbers in patients’ tumors were lower in the group receiving NA therapy prior to surgery; suggesting that CSCs may in fact be sensitive to standard chemotherapy.