Objective To understand the infection of ureaplasma urealyticum in genitourinary of secondary infertility of male and ex-plore the relationship between the genotype of individual ureaplasma species and genitourinary infection of them . Methods Based on the multiple-banded antigen genes (MBA) of ureaplasma urealyticum, 10 pairs of oligonueleotide primers targeting the 5'ends of the MBA genes were designed to identify the MBA genes of U. parvum and U. ureaplasma by PCR-based genotyping system. The 10 pairs of oligonucleotide primers could distinguish the two biovars and 14 serovars of U. ureaplasma. Results A total of 278 (48.6%) positive ureaplasma culture were obtained from 572 patients attending our clinic of reproductive medical eenter. These methods were used to identify and genotype U. ureaplasma in 311 (54.4%) of 572 patients with genitourinary infection among them U. parvum (biovar 1) was detected in 37.1% and U. ureaplasma (biovar 2) in 17.8%. serovar 1 was in 12.4%, serovars3/14 in 17.1% serovar 6 in 7.5%; subtype 1 of biovar 2 was in 5.6%, subtype 2 in 8.9% and subtype 3 in 2.8%, respectively. Conclusion The PCR-based genotyping system will facilitate future stud-ies of relationship between individual Ureaplasma species or subtypes in genitourinary of secondary infertility of male. The methods described here are relatively rapid, practicable, and specific for the detection species identification and subtyping of Ureaplasma species.