Babesia gibsoni and Babesia canis are the etiological agents of canine babesiosis, a protozoal hemolytic disease with global significance. Canine babesiosis has been diagnosed by microscopic identification of intra-erythrocytic trophozoites in blood smear, and by serological testing. Here we developed a quantitative fluorescence resonance energy transfer (FRET)-PCR that amplifies a fragment of the Babesia spp. 18S rRNA gene with high sensitivity and specificity. Melting curve analysis differentiates B. gibsoni, B. canis canis/B. canis vogeli, and B. canis rossi by the disassociation temperature of the fluorescent probes. Babesia gibsoni infection was detected in 8 of 48 canine breeds (17%) and 24 of a total of 235 specimens (10.2%) submitted from 22 states of the continental United States of America. A potential blood donor was positive for B. canis vogeli infection. In Hong Kong (China), B. gibsoni infection was detected in 30 of 64 specimens (46.9%) from 15 of the 24 breeds (63%). While the frequency of canine babesiosis did not associate with seasonal change in Hong Kong, positivity in the USA for Babesia spp. infection was higher in Spring and Summer than in Autumn and Winter. The data suggest that environmental factors associated with tick vector exposure rather than genetic susceptibility determine the incidence of canine babesiosis. Babesia spp. burdens in blood declined significantly with increasing age of the infected dogs, and therapy with atovaquone and tilmicosin eliminated B. gibsoni while doxcycline and berenil did not. This demonstrates that high-resolution real-time PCR analysis may advance diagnosis and therapy monitoring of canine babesiosis.