Unexpected positive cultures (UPCs) are frequently observed in primary shoulder arthroplasty and its clinical significance has not yet been well defined. The aim is to evaluate the UPCs in humeral head in primary shoulder replacement and to understand if UPCs increase in patients with risk factors for contamination (previous surgery or infiltrations). Patients undergoing total shoulder replacement were enrolled in this prospective observational study. To reduce the risk of humeral head contamination, all known procedures to reduce C. acnes burden of the skin were implemented. Patients were divided into 2 groups, namely patients who had undergone previous rotator cuff repair or infiltration and patients with no risk factors for contamination. All the humeral heads harvested were treated with Dithiothreitol, in a specific device (MicroDTTect), to increase the sensitivity of the cultures for bacterial identification. The cultures were analyzed for aerobic and anaerobic bacteria for up to 14 days. The UPCs positivity rate of the 80 patients in the study was 19 % (15 patients). The positivity rates for UPCs in the group with and without risk factors were 30 % (12 patients) and 7.5% (3 patients), respectively. The rate of positive culture was higher in men (87%) than in women (13%). The observed positivity was due to Cutibacterium acnes and peptoniphilus asaccarolyticus, both slow-growing anaerobes. Patients with previous surgery or infiltrations had a 4-fold higher rate of positivity for UPCs compared with patients without previous risk factors. The higher percentage of positivity in patients with risk factors could be related to changes in the joint microenvironment after shoulder procedures. We do not know whether the presence of UPCs could be associated with the development of periprosthetic infections at longer follow-up.