The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PImt) that two maternally unrelated individuals have, by chance, identical SSCP variants varied between 0.14 and 0.30. We detected no heteroplasmy or deviations from strict and stable maternal inheritance when examining four maternal lineages, each represented by six to eight horses, separated by up to five generations from a common ancestral mare. The study establishes a simple screening method for detecting equine mtDNA types, which can be applied for tracing maternal genealogies and for association studies.