Abstract: In order to clarify the genetic diversity and relationship of pathogens of Lasiodiplodia theobromae associated with leaf spot of Ananas comosus from cities and counties in Hainan, the diseased pineapple leaf samples from 16 cities and counties such as Haikou, Chengmai, Danzhou, Sanya and Baoting in Hainan province were used to isolate pathogens. According to the Koch’s rule, 42 pathogens were obtained, then the morphological characteristics were observed, and the genetic diversity was analyzed based on the multiple gene combination sequence. The results showed that 16 pathogens among them were identified as Lasiodiplodia theobromae by morphological characteristics and analysis of phylogenetic tree based on ITS-TUB2 gene combined sequence. The average size of conidia was（22.06～31.07） μm×（11.77～16.48） μm. Then phylogenetic tree based on combined sequences of ITS,TUB-2, ITS, TUB2, EF-1α, GAPDH, CHS-1 and ACT showed that the 16 pathogens were divided into three groups: one mainly concentrated in the middle part of Hainan Island（Danzhou, Changjiang, Baisha, Wuzhishan, Wanning, Qionghai, etc）; one at the north-central part of Hainan Island（Tunchang, Lingao）; the last mainly in the southwest of Hainan Island（Dongfang, Ledong, Sanya, etc）. The results indicated that the genetic diversity of L. theobromae from different pineapple varieties in different producing areas was rich, and it was also clear that L. theobromae was the dominant species in the leaf spot pathogens of pineapple in 16 cities and counties in Hainan province.