Hepatozoon prevalence (occurrence) and parasitemia (intensity) levels were documented in 67 individuals of the threatened gopher tortoise (Gopherus polyphemus) for the first time in four South Florida, US, locations: Jonathan Dickinson State Park (JDSP), Pine Jog Preserve (PJP), Florida Atlantic University Preserve (FAUP), and Blazing Star Preserve (BSP). Sex ratios (males:females) per site were 0.44 at JDSP, 0.72 at PJP, 1.42 at FAUP, and 0.40 at BSP, but no significant differences in the carapace length were found between the two sexes (independent t-test; P=0.101). Hepatozoon sp. was found in 13% (9/67) of tortoises. Percentages of infected tortoises were 22% (5/23) males and 6% (2/33) females. Prevalence and parasitemia were low or nonexistent within sampled tortoises at each of the study sites, although the highest prevalence and parasitemia values were found in gopher tortoises at JDSP (23%, 4/17) which also harbored the highest infection levels, reaching 349/10,000 erythrocytes. No infection was detected within sampled gopher tortoises at PJP.