A seroprevalence study of Anaplasma infection was conducted in a stratified random sample of goats and sheep in Cyprus in which the sample locations were recorded using a geographical information system (GIS). The aim of the study was to estimate the prevalence of Anaplasma phagocytophilum and other Anaplasma species in sheep and goats, and to identify high-risk regions. A total of 689 serum samples (343 from sheep and 346 from goats) were randomly collected and tested for the detection of antibodies against A. phagocytophilum antigen using an indirect immunofluorescent assay. The polymerase chain reaction followed by sequencing analysis was used for the detection and molecular characterization of Anaplasma sp DNA in the blood samples. The prevalence of IgG antibodies against A. phagocytophilum antigen was 18% for goats, and 31% for sheep. Six new genotypes were detected in goats and sheep; by sequence analysis one was identified as A. phagocytophilum, one as Anaplasma platys and the remaining four as Anaplasma species. The results provide evidence for the presence of A. phagocytophilum and Anaplasma species in sheep and goats in Cyprus.