Stem-end rot (SER) caused by Lasiodiplodia theobromae is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC50 values ranged from 0.0006 to 16.4131 µg/ml. A total of 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruits. The mycelial growth, conidia germination and ability to grow at temperatures ranging from 12–35℃ of resistant isolates decreased, suggesting fitness penalties . The experiment showed that after treatment with 10 µg/ml prochloraz, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damages of membranes in the sensitive isolates were more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette subfamily ABCG gene were cloned, but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than those in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.