Tecia solanivora is one of the most damaging pest of potato crops in Central and South America. Eight genetically modified (GM) potato lines with Bacillus thuringiensis cry1Ac gene (Bt potato lines) were characterized at molecular, immunological and biological level under biosafety confined, greenhouse and experimental field conditions. Additionally, preliminary assays regarding substantial equivalence were determined. Cry1Ac protein and gene were detected and quantified on leaf and tubers. DC 40.5, DC 40.7A, DC 40.7B, PP 28, PP 28.1 and PP 40.A, Bt potato lines were resistant to Tecia solanivora when they were exposed. Results obtained indicated that two GM potato lines are promising for further assessments and selection tests as well as future possible commercialization.