Background: Pneumocystis jirovecii primary infection occurs asymptomatically before 6 months of age, suggesting that the infection is acquired very early in life. Furthermore, Pneumocystis pneumonia has been described in newborns, which emphasizes the importance of studying Pneumocystis colonization in mother-infant pairs. Aims: To evaluate the prevalence of Pneumocystis colonization among pregnant women and to determine the potential transplacental transmission. Methods: A cross-sectional study was carried out on HIV-negative women over 18 years-old, and 37 or more weeks of pregnancy attending Hospital Cayetano Heredia Maternity unit during 2016-2017. Clinical and demographical information was collected on them and their newborns. Oropharyngeal washes, nasal swabs, and placenta samples were collected from women, as well as a nasopharyngeal aspirate and nasal swab from newborns. All respiratory samples were analysed by nested-PCR for the detection of Pneumocystis. Placenta samples from women with a positive PCR result in their respiratory samples were also analysed by nested-PCR. Results: Of the 92 pregnant women included, five of them (5.43%) were colonized by Pneumocystis. Pneumocystis DNA was not found in any of the 87 available newborn samples or in the placentas of the five women who had a positive result by PCR in their upper respiratory samples. Conclusions: It was found that 5.43% of the pregnant women were colonized by Pneumocystis, there was no evidence of any role of this colonization in the transmission to their newborns, since none of them tested positive for Pneumocystis.