The tick species, Amblyomma hebraeum and A. variegatum, are the main vectors of Heartwater, a tropical infectious bacterial disease of ruminants caused by Ehrlichia ruminantium. In Mozambique, these tick species have a parapatric distribution, with A. variegatum present in the central and northern regions and A. hebraeum in the South. A narrow overlap area between the distributions of the two species occurs around parallel 22o south. In order to determine the prevalence of E. ruminantium in A. hebraeum and A. variegatum and to determine the genetic diversity and structure of isolates from different localities, adult ticks feeding on cattle and wild ruminants were sampled across the south and center of Mozambique as well as in the adjacent Kruger National Park (KNP), South Africa. The prevalence of E. rumimantium in Amblyomma ticks in relation to the tick species and gender, locality and tick abundance was analyzed. Afterwards, Mozambican Ehrlichia isolates were typed using Multi Locus Sequence Typing and the distribution of groups clustering genotypes were analysed. In total, 722 and 388 of A. hebraeum and A. variegatum ticks were collected from 31 localities and screened for E. ruminantium, using pCS20 nested PCR and Sol1TqM qPCR. The prevalence of E. ruminantium in ticks feeding on cattle varied from 0% to 26.7%, with no infected ticks determined in 7 localities. In ticks feeding on wild ruminants, the prevalence was 8.2 % in the KNP and 6.2% in hunting concessions of the Sofala province. After accounting for the effects of tick gender and sampling sites, no significant difference in prevalence was found between tick species. Most MLST genotypes from Mozambique clustered into subgroup 2C and 2E, which were present in similar proportions in 5 of the 19 localities. Interestingly, MLST genotypes from group G1 and G2D were exclusively found in areas of A. variegatum distribution, while subgroup G2C was only detected in A. hebraeum areas. Moreover, genotypes from subgr