Objective: Obtaining a semen analysis (SA) is an essential step in evaluating infertile men. Despite using standardized procedures for analysis semen quality in the same individual often varies on repeated tests. The objective of this study was to investigate inter-laboratory variation in semen quality between private- and university-based assisted reproductive technology (ART) laboratories.Materials and Methods: IRB approval was obtained to retrospectively evaluate men with a SA at both the private- and university-based ART laboratories. When more than one SA was available from either laboratory, the first at each laboratory was selected for analysis. Comparison of major semen parameters was performed using descriptive statistics and Bland–Altman plots, with differences tested using Wilcoxon-signed rank test.Results: Twenty-eight men aged 33 ± 5 (mean ± SD) years were included in the study. Motility was higher at the private laboratories compared to the university-based laboratory (Median difference −12.5%, 95% confidence interval −20.3%; −5.5%). Percent normal morphology was higher at the university-based laboratory compared to private laboratories (5.0%, 3.6%; 6.9%). No significant differences were found in volume, concentration and total motile sperm count although the Bland-Altman plot bias for concentration was clinically significant (15.9 × 106/ml).Conclusions: In this small series, motility was significantly higher at private laboratories compared to a university-based laboratory but was above WHO reference limits at both places. Normal sperm morphology was significantly lower in semen analyses performed at private laboratories compared to a university-based laboratory and was below WHO reference limits.