Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium (TM2G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium (SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium (MSN), embryo maturation medium (CMM), and shoot elongation medium (CEM), respectively. And the shoots were then rooted on Murashige and Skoog (1962) medium (MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat (SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patts. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.