In this study the generation of kinetoplast DNA (kDNA) probes specific for EthiopianrnLeishmania donovani are described. kDNA was isolated from cultured L. donovanirnpromastigotesand digested with restriction endonuclease. The restriction fragments wererncloned into pUCI8 plasmid vector and propagated in Escherichia coli JMI09 competentrnstrains. Recombinant clones were identified by selecting for plasmid born ampicillinrnresistance followed by colony hybridization screening using radiolabelled L. donovani totalrnkDNA probe. The generated kDNA library was further analyzed by differentialrnhybridizations to total kDNA probes from L. aefhiopica and L. major. Further screeningrnwas conducted by hybridizing radiolabelled plasmid DNA from the L. donovani kDNArnsequence bearing clones with dot blots of L. donovani, L. major, L. aefhiopica and L.rnfropica promoastigotes. Six clones that hybridize only to the parent L. donovani werernselected and analyzed by the same procedure using several L. donovani and other isolates.rnFive clones which hybridize with all Ethiopian L. donovani isolates but not to any of therncutaneous parasites tested, were identified. These L. donovani specific cloned minicirclernkDNA sequences showed various specificities and sensitivities within the L. donovanirncomplex. One of these sequences -Ld 14, specifically detected Ethiopian and other EastrnAfrican visceral Leishmania parasites. A polymerase chain reaction (PCR) assay wasrndesigned for the sensitive detection of visceral Leishmania. The kDNA probes and PCRrndescribed in this study may be useful in the detection of L. donovani parasites in Ethiopia.