To study the genetic character of trispecific hybrid cultivar Shiyuan 321, we analyzed this cultivar and its parents by isozyme and RAPD techniques. The main results are as follows: (1) Esterase isozyme analysis showed that there was one specific band among cultivar Suiyuan 321 and its parents, G. thurberi and G. barbadense; (2) Six specific DNA fragments amplified by 4 primers were found among cultivar Shiyuan 321 and its parents G. barbadense, G. thurberi; (3) By comparing RAPD differences among Shiyuan 321 and its parents, it is indicated that the genetic heterogeneity of cultivar Shiyuan 321 is very high at its genomic DNA level. The above results proved that Shiyuan 321 is a trispecific cultivar from G. barbadense x G. thurberi x G. hirsutum as well as a new germplasm.