The freshwater toxic cyanobacteria Cylindrospermopsis raciborskii CS-505 and Raphidiopsis brookii D9 belong to the order Nostocales of filamentous, heterocystous (N2 fixation) morphotypes. However, Raphidiopsis spp. do not develop heterocysts nor fix N2, contradicting the morphological classification. This work presents an integrated study of evolutionary relationships linked to nitrogen (N) metabolism and toxin production in filamentous cyanobacteria. The genome sequencing and comparison of CS-505 and D9 revealed the smallest genomes of filamentous cyanobacteria and an extent of similarity corresponding to conspecific isolates, therefore recommending a taxonomical reclassification of the genera Raphidiopsis. The presence of heterocyst-specific genes and their expression under alternative N-sources in D9, together with differences in genomic plasticity, allowed us to propose divergent evolutionary pathways for both species. Late transcriptional responses were observed in CS-505 under N-deprivation, contrasting with those of other heterocystous cyanobacteria. The role of repetitive elements and transposases in gene regulation may be critical and requires further investigation.