This work allowed assessing a widespread occurrence of Tychonema bourrellyi in the largest lakes south of the Alps (Garda, Iseo, Como and Maggiore). The taxonomy of the species was confirmed adopting a polyphasic approach, which included microscopic examinations, molecular (16S rRNA and rbcLX sequences) and (Lake Garda) ecological characterisations. Over 70% of the 36 isolates of Tychonema sampled from the four lakes tested positive for the presence of genes implicated in the biosynthesis of anatoxins (anaF and/or anaC) and for the production of anatoxin-a (ATX) and homoanatoxin-a (HTX). A detailed analysis carried out in Lake Garda showed strong ongoing changes in the cyanobacterial community, with populations of Tychonema developing with higher biovolumes compared to the microcystins (MCs) producer Planktothrix rubescens. Moreover, the time × depth distribution of Tychonema was paralleled by a comparable distribution of ATX and HTX. The increasing importance of Tychonema in Lake Garda was also suggested by the opposite trends of ATX and MCs observed since 2009. These results suggest that radical changes are occurring in the largest lakes south of the Alps. Their verification and implications will require to be assessed by extending a complete experimental work to the other large perialpine lakes.