We report here on the development of a nested PCR procedure for the application of Neospora caninum microsatellite markers to clinical samples. Genotyping technology by fluorescently labelled DNA fragment analysis was used in combination with DNA sequencing for those markers which show additional SNPs or complex repetitive sequences. Twenty-nine DNA samples from naturally infected bovine aborted foetuses from two regions of Spain where N. caninum had been detected by nested PCR of the ITS1 rRNA region were analysed using these microsatellites. Complete, or almost complete allele profiles were obtained from 18 samples. Two pairs of DNA samples showed identical profiles, these originated from twins and foetuses from the same herd, respectively. The multilocus analysis performed showed sub clustering of isolates according to their geographical origin. These results highlight the usefulness of these markers for the molecular characterization of isolates of N. caninum and for isolate tracking in live vaccine development.