Histoplasma capsulatum var. farciminosum (HCF), causing Epizootic Lymphangitis (EZL), is endemic in parts of Africa including, Ethiopia, Senegal and Gambia. Despite its high prevalence, impact on animal welfare and socio-economic importance, there is little evidence upon which to build contextually relevant disease control programmes. The performance and availability of diagnostic tests currently used by clinicians is problematic. Methods such as pattern recognition of clinical signs and microscopy lack sensitivity, and other options are either not commercially available or not readily feasible (e.g. culture). This is a significant barrier to further understanding this disease within endemic countries. This study aimed to validate a nested PCR method to confirm the presence of HCF in equine clinical samples. Ethical approval was obtained from the University of Liverpool and the College of Veterinary Medicine and Agriculture. Twenty-nine horses with suspected EZL were included from topographically varied regions of Ethiopia. Clinical examination was recorded and lesion locations drawn onto equine silhouettes. Blood samples and aspirates of pus from unruptured cutaneous nodules were obtained before treatment provided by SPANA. Blood and clinical data were collected from a further 20 horses with no cutaneous EZL lesions. Giemsa stained impression smears of pus and blood were examined microscopically. Aliquots of heat-inactivated pus and blood were inoculated onto Whatman FTA cards and imported to the UK with Defra approved licensing. A nested PCR targeting the ITS region*, was used to identify samples containing HCF, and all PCR products were sequenced. HCF was confirmed in FTA card pus samples from 24 horses, additionally, 23 blood samples were positive from EZL suspected cases. The nested PCR compared favourably overmicroscopic examination of pus, where characteristic HCF yeast bodies were detected in only 14 of the 24 PCR positive samples examined. All nested PCR amplification products were confirmed as Histoplasma spp. by sequencing. Sequencing of cloned PCR amplicons suggested at least two subgroups of HCF based on single nucleotide polymorphisms. These techniques allow the rapid diagnosis of HCF directly from equine clinical samples and offer a useful epidemiological tool. The identification of HCF in blood raises questions about the pathogenesis of HCF in horses and warrants further investigation. *Jiang, B. et. al. (2000) Journal of clinical microbiology 38 (1): 241245. We gratefully acknowledge funding awarded by SPANA UK, the SPANA Ethiopia team for assisting in sampling, and all participating horse-owners. Additional funds were awarded by the Institute of Infection and Global Health, and an SFAM studentship.