Root rot is a major disease of strawberry (Fragaria × ananassa cv. Duchesne) worldwide affecting production and leading to huge economic losses. In 2018 and 2019, strawberry plants displaying root rot symptoms accompanied by plant wilting were observed on commercial farms in Beijing, China. A fungus with the general morphological characters of Neopestalotiopsis was consistently isolated from necrotic lesions in symptomatic roots. Eight representative isolates were selected for further studies. Based on morphological characters and phylogenetic analyses of the combined internal transcribed spacer (ITS) region, partial sequences of the β-tubulin gene (tub2) and the transcription elongation factor 1-α (tef1), all isolates were identified as Neopestalotiopsis rosae. Pathogenicity test showed that N. rosae can produce wilt and root rot symptoms on strawberry plants. Therefore, N. rosae was recognized as the causal agent of root rot of strawberry. This is the first report that discusses the role of N. rosae in causing root rot disease on strawberries in China.