To investigate the pre-vaccination epidemiology of genital human papillomavirus (HPV) infections and genotypes in pregnant Japanese women, we performed Pap smear tests and HPV genotype testing in patients attending Nagasaki University Hospital and collaborating hospitals from August 2007 to July 2010. Serial uterine cervical specimens were obtained from 151 pregnant women. The HPV test was positive on the first visit in 54 women (35.8%; 54/151, average age 30). A total of 49 women (32.5%; 49/151) were infected by at least one high-risk HPV and 5 women were infected by only low-risk HPV. The three most prevalent high-risk HPV genotypes were HPV 52 (31.5%; 17/54), HPV 16 (29.6%; 16/51) and HPV 31 (13.0%; 7/51). The HPV infection pattern (negative, single infection and multiple infection) differed significantly according to the pregnancy trimester (χ(2)-test; P<0.01(Pearson)). Among HPV-infected pregnant Japanese women, HPV52 was the most common genotype. The second most common genotype was HPV16, and these two genotypes accounted for ∼60% of HPV-positive pregnant women. Infection with multiple HPV genotypes was observed more frequently in the first trimester of pregnancy and the pattern of infection changed significantly depending on pregnancy stage.