The highly sensitive nested pCS20 polymerase chain reaction assay for Ehrlichia ruminantium was negative on 506 Amblyomma variegatum from Caribbean islands where clinical heartwater has not been reported, mainly the United States Virgin Islands (18), Dominica (170), Montserrat (5), Nevis (34), St. Kitts (262), and St. Lucia (17). Positive results were obtained with positive controls (Crystal Springs strain) and A. variegatum from countries in Africa where infections are endemic, mainly Tanzania (1/37) and Burkino Faso (2/29). Positive major antigenic protein-1 enzyme-linked immunosorbent assays for E. ruminantium were obtained on convenience samples of sera from apparently healthy cattle, sheep, and goats on Dominica (0/95, 0%; 3/135, 2%; 2/57, 4%), Grenada (0/4, 0%; 1/98, 1%; 1/86, 1%), Montserrat (0/12, 0%; 0/28, 2%; 5/139, 4%), Nevis (0/45, 0%; 0/157, 0%; 0/90, 0%), Puerto Rico (0/422, 0%; 0, 0%), St. Kitts (3/86, 4%; 1/25, 0%; 0/26, 0%), and St. Lucia (0/184, 0%; 0/15, 0%; 0, 0%), respectively. The pCS20 polymerase chain reaction results indicate E. ruminantium is not present on islands where clinical heartwater does not occur. The occasional positive major antigenic protein-1B enzyme-linked immunosorbent assay results appear, then, to be false-positive reactions, and serology appears to be of limited use in testing for E. ruminantium in the Caribbean, as is the case in Africa.