Cytogenetics of triatomines have been a valuable biological tool for the study of evolution, taxonomy, and epidemiology of these vectors of
Trypanosoma cruzi
. Here we present a single microtube protocol that combines micro-centrifugation and micro-spreading, allowing high quality cytogenetic preparations from male gonadal material of
Rhodnius prolixus
and
Triatoma lecticularia
. The amount of cellular scattering can be modulated, which can be useful if small aggregates of synchronous cells are desired. Moreover, a higher number of slides per gonad can be obtained with fully flattened clean chromosomal spreads with minimum overlaps, optimal for classical and modern molecular cytogenetic analyses.
