Pneumocystis jirovecii is an opportunistic fungus predominantly reported in immunocompromised individuals, who develop severe interstitial pneumonia (PcP). However, it is known that asymptomatic or mild pulmonary infections, defined as colonization, are widely observed in the general adult population. So far, genetic and epidemiological data of P. jirovecii infections in Italy are rather scarce and limited to defined geographical regions, mainly regarding isolates from HIV-infected patients. The aim of this study was to evaluate the polymorphisms at the mtLSU-rRNA and the DHPS loci by the screening and genotyping of a cohort of patients from two major hospitals in Rome (Italy). The study included 263 patients divided into two groups, all enrolled consecutively from January 2006 to December 2010: (i) 38 immunocompromised subjects including 25 HIV-infected; (ii) 225 immunocompetent patients. Sixty-seven out of 263 patients (25.5%) were found positive after PCR amplification of the mtLSU-rRNA gene. Overall, genotyping at mtLSU-rRNA locus revealed that the genotype 2 was the most frequent. Sequences of the DHPS gene were obtained from 21 patients, 9 from immunocompromised patients (6 from HIV infected individuals), 12 from immunocompetent ones. Considering the most common DHPS mutations usually detected at amino acid positions 55 and 57 and potentially related to drug resistance, all samples analyzed showed the wild-type signatures. These are the first data in Italy on prevalence and genotypes of P. jirovecii regarding colonized immunocompetent adults. Further multicenter analyses on P. jirovecii infection will be necessary to better define the specific epidemiology of the disease in the Italian populations.