Aim Substantial variation in Gleason grading (GG) of prostate cancer (PCa) exists between Dutch pathology laboratories. This study investigates its impact on treatment strategies. Methods Pathology reports of prostate needle biopsies and clinical data of patients with PCa diagnosed between 2017 and 2019 were retrieved from the Dutch nationwide network and registry of histopathology and cytopathology and The Netherlands Cancer Registry. We investigated the impact of grading variation on treatment strategy for patients whose grade was decisive in treatment choice. First, we evaluated the effect of grading practice (low, average or high grading) on active treatment (AT) versus active surveillance in patients with prostate-specific antigen (PSA) <10 ng/mL and cT1c/cT2a disease. Second, we assessed the association of grading practice with performance of pelvic lymph node dissection (PLND) in patients with PSA 10–20 ng/mL or cT2b disease. We used multivariable logistic regression to analyse the relation between laboratories’ grading practices and AT or PLND. Results We included 30 509 patients. GG was decisive in treatment strategy for 11 925 patients (39%). AT was performed significantly less often in patients diagnosed by laboratories that graded lower than average (OR=0.77, 95% CI 0.68 to 0.88). Conversely, patients received AT significantly more often when diagnosed in high-grading laboratories versus average-grading laboratories (OR=1.21, 95% CI 1.03 to1.43). PLND was performed significantly less often in patients diagnosed by low-grading versus average-grading laboratories (OR=0.66, 95% CI 0.48 to 0.90). Conclusion Our study shows that the odds that a patient undergoes AT or PLND, depends on laboratories’ grading practices in a substantial number of patients. This likely influences patient prognosis and outcome, necessitating standardisation of GG to prevent suboptimal patient outcome.