Evaluation of the possible implication of the SDHA, SDHB, SDHC, SDHD and CS genes in non-obstructive male infertility was performed on the basis that sperm concentration in the ejaculate has been previously correlated with nuclear-encoded mitochondrial enzyme activities (the four subunits of succinate dehydrogenase/complex II of the respiratory chain and citrate synthase). We performed an exhaustive analysis of the five genes for the presence of sequence variants that could be associated with impairment of sperm production. blastn searches in the genomic sequence NCBI database evidenced the presence of highly homologous sequences elsewhere on the genome that can interfere with polymerase chain reaction experiments. Therefore, a careful design of the analytical strategy to search for sequence variants was performed. In this report, we provide primer sequences that allowed selective amplification of coding and immediate flanking regions of the five genes. Fifty-five sequence variations in the five genes were identified in infertile and normozoospermic fertile individuals as controls and only one of them (SDHA c.456+32G>A) showed significant genotype association with impairment of sperm production. Moreover, new single nucleotide polymorphisms identified should be useful in future association studies for other human diseases related to nuclear-encoded genes, leading to mitochondrial respiratory chain activity impairment revealing the physiological role of these genes.