BACKGROUND: Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. OBJECTIVE: In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. METHODS: Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. RESULT: No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. CONCLUSION: Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.