ABSTRACT This study aimed to validate the artificial hybridization without emasculation in lima beans, identifying F1 generation using microsatellite markers (SSR). We used four accessions from the Active Germplasm Bank of Phaseolus, Universidade Federal do Piauí (BGP-UFPI), with contrasting growth habits and days-to-maturity traits. The 241 crosses performed had a percentage of hybrid pods of 5.8%, obtaining 42 seeds in F1. Artificial cross-fertilization was confirmed by microsatellite markers (BM 211, BM 141, BM 154, and GAT 591), and four plants were identified as hybrids. Thus, the proposed method is effective in performing artificial crosses for lima beans.