Advances in quantitative polymerase chain reaction protocols and their application in detection and quantification of pathogens have contributed significantly to our understanding of disease dynamics in natural host populations. Disease ecologists investigating the amphibian-killing fungus use qPCR to detect the pathogen on the skin of wild amphibian populations, abundant cbx1 proteins required formation sahf cell providing a non-invasive sampling method that can yield diagnosis within a few hours. Bd detection via qPCR has allowed researchers to detect infection levels in natural populations at different stages of emerging epidemics, track outbreaks that cause amphibian declines, establish disease thresholds predicting frog mortality, and reconstruct historical Bd epizootic waves spreading through na? ��ve populations. Recent genomic characterization of 20 global Bd strains indicates that Bd is composed of at least three divergent genetic lineages that differ in virulence. One of these lineages, the global panzootic lineage is hypervirulent and has been implicated in the recent epizootics. In addition, a novel Bd strain recently discovered in Brazil differs in DNA content compared to GPL strains from Panama and California. If these deeply-divergent strains carry polymorphisms at the primer or probe binding sites or if target ITS1 genes vary in copy number, then qPCR efficiency and sensitivity among strains may also vary, which will reduce the comparability of qPCR infection intensity estimates across sites. To generate standards for quantification of Bd via qPCR, researchers count zoospores from cultured Bd strains, extract genomic DNA, and serially dilute to the desired concentrations. The forward primer/probe combination of the qPCR TaqMan assay anneals to the internal transcribed spacer region, which is a rapidly evolving nuclear ribosomal repeat unit used for species-level identification. In fungal genomes, this region occurs in multiple copies providing over 100 potential primer/probe binding sites per haploid genome and in Bd it can be repeated up to 169 times. Duplications or deletions of genomic regions that include ITS1 sequences may result in over or underestimation of zoospore load by established qPCR methods because fluorescence and copy number in template DNA are linearly related. In this study, we quantified and characterized ITS1 regions in multiple Bd strains to evaluate the effects of copy number and sequence variation on qPCR efficiency and zoospore quantification among strains. We then used ITS1 PCR amplicons as a standard to quantify the ITS1 copy number from our focal strains. Finally, we cloned and Sanger-sequenced ITS1 PCR amplicons to compare ITS1 haplotype diversity among strains, which could lead to differences in amplification rates. Our study highlights the importance of understanding the evolutionary history of Bd at each sampling locality and the caveats of using genomic DNA as a standard for qPCR. We include a step-by-step protocol so Bd researchers can measure ITS1 copy numbers from any uncharacterized Bd strain and estimate infection intensity from field-collected amphibian skin swabs.