Ecuadorean mashua (Tropaeolum tuberosum) germplasm has been found to be widely infected with the potyvirus Tropaeolum mosaic virus (TropMV). The objective of this research was to produce virus-tested (VT) germplasm to compare growth in vitro and in the field with virus-infected (V) germplasm. Twenty-three of 25 apical dome-derived clones tested free of virus based on bioassays using Nicotiana benthamiana and Chenopodium quinoa. In vitro-generated plant tissue was just as effective for determining VT status as greenhouse-generated plant tissue. Genotype rather than virus-infection status appeared to have a greater effect on in vitro proliferation. There were no differences in in vitro rooting among the genotypes or between the VT clones compared to the V clones, with at least 90% of the microcuttings rooting. However, rooted microcuttings of V clones were taller than rooted microcuttings of VT clones. Plants were readily re-established in a greenhouse at the USDA, Foreign Disease-Weed Science Research Unit at Fort Detrick, USA. In field experiment 1, ca. 75% of the plants survived field transplanting and VT plants of genotype 1147 had greater tuber weight (928 g) than V plants (235 g). In field experiment 2, plant mortality was high one month after field transplanting. Genotypes 1093 (59%) and 1141 (54%) had higher survival than genotype 1147 (44%); however, survival did not differ between the VT (46%) and V (59%) plants of all genotypes. No differences were noted in field performance for the three genotypes after 10 months of growth. Although overall tuber yield among the V, VT and VTR (reinfected-VT plants) did not differ, V plants produced big tubers that weighed more than those from VT plants. Thirty-three percent of the VT plants became reinfected and 42% of the V plants tested negative after 10 months in the field based on double-antibody-sandwich ELISA.