Presently used techniques for making transgenic animals are cumbersome; require trained man-power, costly infrastructure and large number of zygotes at the expense of several females. The ability of the male germ cells to integrate foreign genes provides opportunity for developing alternate methods for generation of transgenic animals. We have developed a reproducible protocol for transfecting mammalian and non-mammalian genes in repopulating undifferentiated spermatogonial cells through in vivo electroporation of the testis. More than 90% of the male mice electroporated with transgene of choice sire transgenic pups upon mating with the wild type (WT) females. Such males serve as permanent resource for the production of transgenic founders. This technique has several advantages over the presently used techniques including drastic reduction in the time required and in the usage of animals for generation of transgenic progeny. Hence, this protocol is likely to be applicable to many species.