Several case-control studies have investigated the effect of CAG repeat length variation in the POLG gene on male fertility and semen quality. Some described an association between the homozygous not10 CAG-repeat genotype and male subfertility and/or reduced semen quality, whereas others did not. The aim of our study was to investigate whether the not10/not10 variant is associated with spermatogenic failure. By direct sequencing methods, we determined the CAG repeat length of POLG in a cohort of 700 consecutive included men with variable degrees of spermatogenesis to investigate its effect on semen quality. The frequency of the not10/not10 variant in our cohort was 4.7%. There were no differences in semen quality between groups with various POLG genotypes. There was a significant difference in frequency of the three CAG-repeat genotypes between ethnic subgroups. In conclusion, the not10/not10 POLG variant is not associated with clinically significant decreases in semen quality, but its frequency is dependent on ethnic background.