Pneumocystis species are obligate fungi capable of causing opportunistic infection called Pneumocystis Pneumonia (PCP) in those who are immunocompromised, often resulting in serious disease and a high mortality rate. Due to host specificity and inability to conduct in vitro studies, molecular methods have stepped in to undertake the bulk of epidemiological studies. Many unknows regarding the fungus still exist, and there is little concordance between researchers. This thesis aimed to improve and standardize the identification and genotyping techniques currently used to enable the global research community to bridge gaps collaboratively. Chapter 1 outlines Pneumocystis epidemiology, the pitfalls facing researchers and how they have been addressed. As a major influencing factor in determining sequencing success is the quality of the DNA obtained, Chapter 2 investigates and compares several DNA extraction kits and methods for an effective protocol. The gold standard for P. jirovecii currently remains to be multilocus sequence typing (MLST), therefore Chapter 3 optimizes primers and PCR protocols of the current MLST scheme used by the International Society for Human & Animal Mycology. It identified pitfalls within the scheme and addresses them by reviewing all genotyping loci and schemes used till now to determine the most effective scheme to be used in the future. This resulted in the proposal of a new global consensus MLST scheme, which was then successfully applied to a clinical cohort in Chapter 4. Chapter 5 examines the epidemiology of globally collected clinical samples using the scheme, uncovering unique alleles that formed 49 sequence types, and demonstrated the importance of a collaborative database. The importance of the genetic locus DHPS to monitor global resistance is discussed in Chapter 6. Finally, long-read next-generation sequencing using the MinION sequencer is applied to detect PCP in clinical samples to determine its potential for early diagnostics of PCP in Chapter 7.