To investigate the inheritance of the mitochondrial(mt)DNA in the interspecific crossing of Genus Cucumis, fragments of the cob, nad1, nad7 mt gene were amplified and sequenced from S5 progenies of allotetraploid species(Cucumis hytivus Chen and Kirkbride.), cultivated cucumber ‘BeijingJietou’ (C. sativus L.) the paternal parents and the wild Cucumis species (C. hystrix Chakr.) the maternal parents, respectively. One, seven and seventeen polymorphic loci’s in the corresponding 909 bp cob, 943 bp nad1 and 880 bp nad 7 gene among three Cucumis species were detected, respectively. Among them, one, six and fourteen polymorphic loci’s in the mt gene of allotetraploid were revealed to be identical as the cultivated cucumber, the paternal parents, but different from the wild species , the maternal parents; the rest one and three polymorphic loci’s were found to be different from both parents. This result demonstrated that polymorphic loci’s in the mt DNA of S5 allotetraploid, the hybrid progeny, were dominantly transmitted from paternal parents but not maternal parents in the hystrix譻ativus interspecific crossing, and the mt DNA was dominantly paternal transmission.