A total of 707 isolates of Botrytis were collected from plastic tunnel-grown strawberry and tomato in Hubei province of China. They were identified based on the specific molecular markers. Diversity of the B. cinerea (Bc) isolates was evaluated by typing the transposable elements or TE (Boty, Flipper) and the mating types (MAT1-1, MAT1-2), as well as by determining virulence on tobacco (Nicotiana benthamiana) and fenhexamid sensitivity in agar medium. The results showed that 706 isolates (accounting for 99.9%) belonged to Bc, one isolate (accounting for 0.1%) belonged to B. pseudocinerea. The Bc isolates fell into four TE types, Vacuma, Boty, Flipper and Transposa, which accounted for 3.1%, 9.6%, 18.4% and 68.8% (n = 706), respectively. The strawberry and tomato sub-populations of Bc differed significantly (P < 0.05) in composition of the four TE types. The overall ratio of MAT1-1 to MAT1-2 deviated from 1:1 (n = 706, P = 0.0002), MAT1-2 (accounting for 56.9%) predominated over MAT1-1 (accounting for 43.1%). However, in 7 out of 12 geographic sub-populations, the ratio of MAT1-1 to MAT1-2 matched 1:1, whereas in the remaining five geographic sub-populations, the ratio of MAT1-1 to MAT1-2 did not match 1:1. Results of the biological characterizations showed that most Bc isolates were highly sensitive or sensitive to fenhexamid, and the majority of Bc isolates were highly virulent or virulent on tobacco. Moreover, relationship between genetic diversity and biological characteristics was analyzed. The results achieved in this study are helpful for understanding of the populations of B. cinerea.