Background: Preoperative bladder urine culture (PBUC) analysis has become a standard application before any stone surgery. When growth is detected in PBUC, it is contraindicated to perform flexible ureterorenoscopy (f-URS). The results of the PBUC susceptibility test do not correlate well with those of the renal pelvic urine culture (RPUC) analysis. Previous studies have demonstrated the positivity of RPUC as an important marker for the development of infections after endoscopic operations. In the current study, we aimed to evaluate the consistency between PBUC and RPUC and to identify preoperative markers associated with a positive RPUC. Methods: Data from 129 patients who underwent f-URS on renal and proximal ureteral stones in two centers between 2015 and 2020 were prospectively recorded in a database and retrospectively analyzed. PBUC was obtained from all the patients preoperatively, and RPUC was taken at the beginning of the f-URS operation. The results of the two cultures were compared. Results: There was growth in PBUC in 25 (19.4%) patients and RPUC in 35 (27.1%) patients. Possible predictive markers in predicting a positive RPUC were evaluated using multivariate logistic regression analysis. Preoperative urine density at the renal pelvis [odds ratio (OR): 0.848, p<0.001],grade≥2 hydronephrosis (OR:18.970,p=0.001), and lower calyceal stone localization (OR:0.033,p=0.017) were determined as independent predictive factors for a positive RPUC. The ability of pelvis urine density to predict positive RPUC positivity was evaluated using the receiver operating characteristic analysis, in which the area under the curve value was determined to be 0.858 (0.780-0.936). The cut-off value of pelvis urine density in the prediction of RPUC positivity was 4.5, at which it had 80% sensitivity and 77.7% specificity. Conclusions: PBUC may not represent true colonization. Although bladder urine culture is negative before the operation, patients with preoperative hydronephrosis and low pelvis urine density may have RPUC growth.