This study analyzed microsatellite distribution in the holocentric chromosomes of Triatominae, Chagas disease vectors. Using a non-denaturing FISH technique, 16 microsatellites were examined across 25 species from the Triatomini and Rhodniini tribes. Three hybridization patterns emerged: strong signals in specific regions, dispersed signals based on microsatellite abundance and the absence of signals in certain regions or chromosomes. Rhodniini had weak and scattered signals, indicating low microsatellite abundance, while Triatomini showed diverse and abundant patterns, suggesting their significance in genomes. Particularly, all Triatomini species exhibited a high abundance of GATA repeats in the Y chromosome, unlike Rhodniini. This suggests the ancestral trait is specific to Triatomini. The study provides insights into microsatellite composition and distribution in Triatominae genomes, shedding light on their evolutionary processes and relationships with other reduviid groups.