Fifty-two febrile patients living in Barquisimeto, Venezuela, were screened for arbovirus infection by virus culture during an outbreak of what was thought to be Zika virus infection. We report identification of Mayaro virus (MAYV) on culture of plasma from one patient, an 18-year-old woman with acute febrile illness, arthralgias, and psoriasiform rash. The strain was sequenced and was found to be most closely related to a 1999 strain from French Guiana, which, in turn, was related to two 2014 strains from Haiti. By contrast, previously reported outbreak-related MAYV strains from a sylvatic area approximately 80 miles from where the case patient lived were most closely related to Peruvian isolates. The two strain groups show evidence of having diverged genetically approximately 100 years ago.