Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immuno-compromised patients. To analyze the genetic diversity of Pneumocystis jirovecii in HIV-negative patients in China, respiratory specimens were obtained from 105 patients who tested PCR-positive for the presence of the Pneumocystis jirovecii mitochondrial large subunit ribosomal RNA (mtLSU rRNA) between 2011 and 2013. Pneumocystis jirovecii isolates were genotyped based on the upstream conserved sequence (UCS) of the major surface glycoprotein (MSG) gene and the internal transcribed spacer (ITS) of nuclear rRNA operon. Eighty-one of the 105 isolates showed a positive PCR for the UCS region. We identified six different patterns comprising two, three, four, or five UCS repeats, including 1, 2, 3 (69.14%), 1, 2, 3, 3 (22.22%), 1, 2 (3.7%), 1, 1, (2.47%), 2, 2, 3, 3 (1.23%), and I, I, 2, 3, 3 (1.23%). In regard to the ITS region, 58 of the 105 isolates were cloned and sequenced successfully. Six known ITS1 alleles (A, B, DEL1, E, N, and SYD1), two new alleles (designated as BTM3 and BTM4), six known ITS2 alleles (a, b, i, g, h and O) and one new allele (designated as btm6) were observed. A total of 19 Pneumocystis jirovecii ITS haplotypes were identified. The most frequent type was Bi (25.9%), followed by Ai (13.8%), Eb (10.3%), and SYD1 g (6.9%). Among the 58 specimens examined, 49 (84.5%) were found to contain a single type of Pneumocystis jirovecii, while 9 (15.5%) contained multiple genotypes. A total of 34 allelic profiles were observed in 58 isolates when the two loci were combined with each other. A Fisher's exact test revealed that there was no statistically significant (P = 0.330) association between the most frequent UCS and ITS genotypes. An analysis of the phylogenetic relationship between different patient groups identified two major groups based on the sequence variations of concatenated UCS and ITS sequences in 49 isolates. Our results demonstrated the high genetic variability of Pneumocystis jirovecii in HIV-negative patients in China. (C) 2015 Published by Elsevier B.V.