Genetic analysis has demonstrated distinct variation in the rDNA internal transcribed spacer 2 sequence in 2 populations of Amblyomma americanum (colony and wild). The ability to transmit the protozoan parasite Theileria cervi was quantitatively assessed of for each of these 2 genetically characterized populations of A. americanum. Feeding success of individuals of both populations of A. americanum was compared using the percentage tick yield and engorgement weight of acquisition-fed nymphs. Vector competency of individuals within both populations of A. americanum was measured using the prevalence, abundance, and intensity of infection as defined by the number of T. cervi–infected acini. Significantly higher proportions of acquisition-fed nymphs of colony and wild A. americanum were recovered, depending on which donor deer they infested. A significant correlation was not detected between percentage tick yield and parasitemia of donor deer. Wild A. americanum nymphs had significantly higher engorgement weights than colony ticks; however, a negative correlation was observed between the average T. cervi parasitemia of donors during acquisition feeding and the engorgement weight of wild A. americanum. Significant differences were not detected in the prevalence or mean abundance of T. cervi–infected A. americanum adults. Female A. americanum were more heavily infected with T. cervi than male ticks. A significantly higher proportion of wild female A. americanum were more heavily infected (>170 infected acini per infected tick) with T. cervi than colony female ticks. These findings support the hypothesis that genetically distinct populations of ticks can vary in their ability to acquire, maintain, and perhaps transmit infectious agents. The influence that variation in vector competency of ticks has on the occurrence, distribution, and risk factors related to newly recognized or emerging tick-borne diseases of domestic and wild animals has yet to be determined.