Crown rust of barley, caused by Puccinia coronata var. hordei, was first reported by Jin and Steffenson in 1992, and the fungus has been reported only in the United States and Hungary. In China, stripe, stem, and leaf rusts have been reported on barley, but not for crown rust. Recently, a sample (HZJ0004) of rust collected from barley in Qilian county in Qinghai, China, appeared different from the three rusts based on color, size, arrangements of uredinia and/or telia. Teliospores had crown-shaped appendages on the top. Based on the disease symptoms and morphology of urediniospores and teliospores, the fungus was identified as P. coronata var. hordei. Using the internal transcribed spacer sequences, the isolates HZJ0004 from barley and POR3 from buckthorn (Rhamnus sp.) were clustered in one clade with P. coronata var. hordei isolates from barley and Elymus repens but in a different clade from the isolate POC8 from wild oat and the varieties of P. coronata from oats and grasses. At the seedling stage, most of the tested cultivars of barley and rye were susceptible to P. coronata var. hordei isolates HZJ0004 and POR3, but the cultivars of oats, triticale, wheat, and most grasses of genera Aegilops, Brachypodium, Bromus, Calamagrostis, Deschampsia, Elymus, Festuca, and Phleum were resistant, indicating their host specialization on barley. To our knowledge, this is the first report of crown rust on barley in China.