Ehrlichia ruminantium, the etiologic agent of the economically important disease heartwater, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, particularly A. hebraeum and A. variegatum. Although serologic and microscopic evidence of the presence of heartwater have been reported in ruminants in Cameroon, knowledge of E. ruminantium infection in the tick vector, A. variegatum, is lacking. In order to determine the infectivity of A. variegatum ticks by E. ruminantium, we analysed 500 un-engorged A. variegatum ticks collected by hand-picking from predilection sites from 182 cattle [115 ticks from 82 cattle at Société de Développement et d'Exploitation des Productions Animales (SODEPA) Dumbo ranch (SDR) and 385 ticks from 100 cattle at the Upper Farms ranch (UFR)] by amplification of the open reading frame (ORF) 2 of the pCS20 region of E. ruminantium. PCR amplification of the 279 bp fragment of the pCS20 region detected E. ruminantium DNA in 142 (28.4 %) of the 500 ticks with a higher infection rate (47/115; 40.9 %) observed in ticks from SDR and 24.7 % (95/385) of ticks collected from cattle at UFR. Twenty five randomly selected PCR products were sequenced and results indicated that some of the isolates shared homology with one another and to sequences of E. ruminantium in the GenBank. This report represents the first molecular evidence of E. ruminantium infection in A. variegatum ticks in Cameroon and suggests possible exposure of cattle to this pathogen in our environment.