This study describes the invasion of the upper Paraná River basin by Pterygoplichthys ambrosettii based on a literature review and field samples. Pterygoplichthys ambrosettii has been reported in 42 localities throughout the upper Paraná River basin, including the Tietê, Paranapanema, Paraná, Grande and Aguapeí rivers. The ascent of P. ambrosettii after the inundation of the Sete Quedas Falls on the Paraná River and the release of individuals by aquarium hobbyists were the primary drivers of this invasion.