At present, there are more than one hundred scientific name all over the world and about twenty interiorly of Pleioblastus (Bambusoideae ). Because of its graceful form, Pleioblastus can be used as landscaping and garden landscape, much more attention has been paid to its ecological and economical values. With the rapid development of modern molecular biology techniques, the molecular marker technology has widely been used in the aspect of bamboo taxomomy and genetic diversity, such as assistant classification at the molecular level, which is in favor of solving classification disputes and important to identify the germplasm resources. Recently, some Pleioblastu classifications have been quite controversial in the academic field, and just a few researches about applying molecular marker technology to genetic relationships in bamboo speciess have been covered. As is known to all, ISSR (Inter simple sequence repeats) is proposed by Zietkiewicz, better at stability and polymorphism, easy to operate, with low technical requirements and cost, and the ideal molecular marker technology to construct DNA fingerprinting. Currently, ISSR has been widely used to the identification of varieties, analysis of genetic diversity and so on. Utilizing 18 screened primers with better polymorphism on bamboo and optimized ISSR reaction system and amplification program in this paper, the genetic diversities of twenty-five kinds of bamboo in Pleioblastus were analyzed, including P.china f. hisauchii, P.gramineus (Bean) Nakai, P.amarus (Keng) Keng f. var. pendulifolius S.Y.Chen, P.amarus (Keng) Keng f. var. subglabratus S.Y.Chen, P.qingyuanensis, P.chino f. angustifolius, P.suichangensis, P.simonii f. albostriatus, P.longifimbriatus S.Y.Chen, P.dokgyoamus, P.oleosus Wen, P.amarus var. (Keng) Keng f. var. hangzhouensis S. L. Chen et S. Y. Chen (accepted name), P.amarus (Keng) Keng f., P.kongosanensis f. aureostriaus, P.gozadakensis Nakai, P.hsienchuensis Wen, P.yunhoensis, P.maculates (McClure) C.D.Chu et C.S.Chao, P.juxianensis Wen, P.intermedius S.Y.Chen, P.maculosoides Wen, P.yixingensis S.L.Chen et, P.longifimbriatus S.Y.Chen, P.graminens f. manstopiral, P.rugatus Wen et S.Y.Chen. The results showed that Eighteen ISSR primers amplified 155 DNA fragments, of which 137 (88.3%) showed polymorphic with good repeatability among the populations, which was to say "each primer amplified 8.61". The DNA molecular weight was from 160 bp to 3000 bp. The average genetic distance of twenty-five kinds of bamboo in Pleioblastus was 0.5006, and the variation of its genetic distance was from 0.1486 to 0.7191, which indicated that the Pleioblastus were in a high genetic diversity, and relatively complex interspecific genetic. At the same time, ISSR cluster analysis revealled that the bamboos were divided into three categories at genetic distance 0.5396, and the results were generally consistent with the morphological classification, which illustrated that ISSR technology detected accurately genetic diversity and genetic relationship of some plants in Pleioblastus, to contribute to the classification of the genus. In the study, digital fingerprint identification code of twenty-five bamboos in Pleioblastus were built with 7 primers (U807, U815, U835, U836, U840, U841, U844), it will provide reference for classification and gerplasm identification of some plants in Pleioblastus.