Horizontal gene transfer is a fundamental strategy for prokaryotic adaptation and surveillance, so improving the knowledge in this field could turn in a strategy for the exploitation and improve of biological process applied to the industry. In the extremophile bioleaching bacterium Acidithiobacillus ferrooxidans there is a 300 Kbp integrative and conjugative element that is active in excision, and inserts in a tDNA Ala gene in the genome. Among species from the genus Acidithiobacillus there is a common genetic context around the tDNA target for the integration, and presents a high similarity between the integrases, the enzymes that catalyze the process. Through DNA – protein binding assays and the developing of a genetic assay for in vivo integration, this work determined the influence of the genetic context in the choice for the integration site, and investigate whether these similarities at DNA and protein level allowed the integration of an integrative and conjugative element between two species of the genus. Our findings show an independence of the genetic context in the site-specific integration and demonstrate the high selectivity of the integrases despite their similarities.PFCHA-BecasPFCHA-Becas