At low trophic state, stable stratified water columns may provide favorable conditions for adapted phytoplankton species to form deep chlorophyll maxima (DCM). Such maxima occur regularly in Lake Stechlin, mainly contributed by the cyanobacterial species Cyanobium sp. and occasionally by Planktothrix rubescens. In the early twenty-first century, a rapid invasion by nostocalean cyanobacteria occurred in the lake and a number of Dolichospermum species together with Aphanizomenon flos-aquae appeared. As revealed by both microscopic and fluorimetric methods, during the summer stratification of 2013, a multispecific DCM was formed by Cyanobium, Planktothrix rubescens, and A. flos-aquae, however with spatial segregation. Planktothrix occurred in the upper hypolimnion, Aphanizomenon and Cyanobium dominated in the metalimnetic layer. Coexistence of these three cyanoprokaryota is possibly the consequence of different environmental factors limiting them (light, availability of N and P). This study represents a rare case when spatial niche segregation of phytoplankton species occurs in close to equilibrium conditions. DCM formed by Aphanizomenon and Cyanobium was detected by the fluoroprobe; Planktothrix with its different pigment compositions remained largely hidden. Our results indicate the necessity of parallel microscopic investigations and the need of careful calibration when fluorimetric methods are used for detecting cyanobacterial populations.