This technology includes isolate of Middle East respiratory syndrome coronavirus (MERS-CoV) for antiviral and diagnostic development. The isolate was confirmed by full genome sequencing and further characterized by serologic and molecular testing and electron-microscopical studies. This is the first MERS-CoV strain isolated from a US imported case of MERS. The isolation of this virus underscores the importance of combing classic techniques such as virus isolation and electron microscopy and molecular techniques, such as real-time PCR and full genome sequencing, in identifying etiologic pathogens.