Abstract The zoonotic protozoan Cryptosporidium parvum causes around half of cryptosporidiosis cases in the UK. Sexual recombination drives genetic variation and merits a multilocus genotyping (MLG) approach to track sources of contamination and routes of transmission, and inform risk assessments and outbreak investigations. To encourage standardisation across countries and between laboratories with varying resources, we used whole genome sequencing to identify new variable numbers of tandem repeat markers, predicted to be suitable for fragment sizing by capillary electrophoresis. Seven of 28 markers identified initially in the C. parvum reference genome (Tandem Repeats Finder, New York) were selected on the basis of un-linked, simple tandem repeat motifs of ≥6 bp displying variable numbers in in-house genomes. Four were 6 bp motifs and the others 12, 15 and 27. PCR primers were designed and fragment sizes estimated for 20 C. parvum samples using a QIAxcel Advanced (Qiagen) machine, run under optimised conditions, and compared with sequencing. Estimated fragment sizes were confirmed in most samples. When repeatability and reproducibility was investigated using triplicate PCR reactions of four samples on two occasions, discrepancies were observed in a small number of reactions but were resolved readily. However, when applied to a larger validation panel of 269 C. parvum samples representing spatio-temporal variation, outbreaks and sporadic infections of humans and animals, 5% PCR reactions were not detectable, and a further 5% provided inconsistent and unresolvable ambiguous sizes especially in the 6 bp repeat markers. To improve accuracy and reliability, we re-evaluated the MLG scheme using labelled primers for fragment sizing on a different platform (3500 Genetic Analyzer, Applied Biosystems™). Multiplexing the PCRs provided an economic approach, and the sample archive held at the reference unit enabled full scale validation (typability, discriminatory power, epidemiological concordance and efficiency) of a MLG scheme for C. parvum. Funding EU Framework Seven 'Aquavalens'