Background Yacon (Smallanthus sonchifolius) is a root crop native to the Andean region. Low sexual reproductive capacity is a major constraint facing the genetic breeding of this crop. Biotechnological techniques offer alternative ways to widen genetic variability. We investigated somaclonal variation in regenerants of yacon derived from in vitro somatic embryogenesis using simple sequence repeat (ISSR) analysis and flow cytometry. Results Twenty tested ISSR primers provided a total of 7848 bands in 60 in vitro regenerants and control plant. The number of bands for each primer varied from 3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. ISSR analysis revealed genetic variability in 6 plants under study. These regenerants had Jaccard's distances 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040. Flow cytometric analysis did not reveal changes of relative nuclear DNA content in regenerants suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid levels. Conclusions Our findings show that indirect somatic embryogenesis could be used in yacon improvement to widen genetic variability, especially when low sexual reproductive capacity hinders classical ways of breeding.