We have developed and evaluated a real-time reverse transcription PCR (RT-PCR) assay for detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak associated with severe respiratory illness in the United States. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, Missouri. This sequence, along with other GenBank® sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP-1 gene with an analytic limit of detection of 4 copies per reaction, and was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested, and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.