Canine babesiosis is an important worldwide disease caused by protozoan hemoparasites of the genus Babesia, which are primarily transmitted to a dog by the bite of an Ixodid tick, although vertical transmission has recently been reported. The disease is typically characterized by hemolytic anemia, fever, splenomegaly, and thrombocytopenia, with clinical signs ranging from clinically normal to acute anemia. Death may even result in some severe cases. Two species of Babesia, Babesia gibsoni and Babesia canis, have long been known to cause babesiosis in dogs. To date, almost all B. gibsoni infections in the United States have been reported in American Pit Bull Terriers or in dogs associated with the breed through either transfusion or fighting. Dog blood samples received from kennels, shelters, and veterinary clinics throughout Texas were tested for the presence of B. gibsoni and B. canis. A total of 254 samples were tested for B. gibsoni and B. canis by light microscopy and polymerase chain reaction (PCR). Babesia gibsoni was detected in four of the dogs tested and B. canis was detected in one of the dogs tested. The average packed cell volumes (PCVs) of infected dogs were compared with those of uninfected dogs, with the infected, on average, having lower PCVs. Molecular characterization of the small subunit ribosomal RNA gene and the ribosomal RNA internal transcribed spacer regions was performed on all sequences obtained in this study, and results were consistent with those previously reported for B. gibsoni and B. canis. Also, positive samples and additional samples provided by North Carolinia State University were used to initiate in vitro cultures of the parasites. To date, one isolate of a large unknown Babesia sp. from a North Carolina dog was successfully established in vitro. The establishment of Babesia spp. parasites in culture may aid in the development of a vaccine for babesiosis and will also be beneficial in improving diagnostic tests for the parasite.