In May 2015, Zika virus was reported to be circulating in Brazil. This was the first identified introduction of the virus in the Region of the Americas. Since that time, Zika virus has rapidly spread throughout the region. As of April 20, 2016, the Florida Department of Health Bureau of Public Health Laboratories (BPHL) has tested specimens from 913 persons who met state criteria for Zika virus testing. Among these 913 persons, 91 met confirmed or probable Zika virus disease case criteria and all cases were travel-associated (1). On the basis of previous small case studies reporting real time reverse-transcription polymerase chain reaction (RT-PCR) detection of Zika virus RNA in urine, saliva, and semen (2-6), the Florida Department of Health collected multiple specimen types from persons with suspected Zika virus disease. Test results were evaluated by specimen type and number of days after symptom onset to determine the most sensitive and efficient testing algorithm for acute Zika virus disease. Urine specimens were collected from 70 patients with suspected Zika virus disease from zero to 20 days after symptom onset. Of these, 65 (93%) tested positive for Zika virus RNA by RT-PCR. Results for 95% (52/55) of urine specimens collected from persons within 5 days of symptom onset tested positive by RT-PCR; only 56% (31/55) of serum specimens collected on the same date tested positive by RT-PCR. Results for 82% (9/11) of urine specimens collected >5 days after symptom onset tested positive by RT-PCR; none of the RT-PCR tests for serum specimens were positive. No cases had results that were exclusively positive by RT-PCR testing of saliva. BPHL testing results suggest urine might be the preferred specimen type to identify acute Zika virus disease.