Multilocus sequence typing (MLST) is considered the preferred approach for the analysis of genetic diversity of P. jirovecii, but no consensus scheme is currently established. Previous research compared all MLST schemes currently used worldwide, to determine a MLST scheme that would combined loci with the highest amplification efficiency and discriminatory power. Based on the analysis of available sequences CYB, SOD, mt26s were identified to produce the most discriminatory MLST scheme for P. jirovecii genotyping. The herein presented study looked at the efficiency of the scheme in a real-life setting and assessed whether the scheme could be used for all PCP infected animal isolates, as well as a world-wide standardized MLST scheme for all Pneumocystis species. The newly identified MLST loci (CYB, SOD and mt26s) were amplified and sequenced from 30 clinical PCP isolates and 2 canine samples. In addition the DNA concentration needed for a successful amplification was assessed. The CYB, SOD and mt26s scheme successfully generated MLST data from 28/30 PCP isolates, even with minimal DNA concentrations. It was noted that no MLST sequence data were obtained if the DNA concentration was diluted below 20 ug/ml. Further optimization to the protocol is currently underway to ensure higher amplification rates. More mammal isolates over a larger range of animal species is required to confidently recommend a universal consensus scheme for all Pneumocystis.