The diversity and ecological relevance of small haptophytes in marine systems is increasingly recognized. Similar investigations in freshwater remain scarce, despite some recent studies showing the existence of divergent haptophyte lineages and indicating that these microalgae can occur at high abundance in lakes. We studied the diversity of haptophytes in a wide variety of marine, salty continental and, most particularly, freshwater environments by amplifying, cloning and sequencing 18S rRNA genes. For this purpose, we designed two sets of primers specific for the two recognized haptophyte classes, Prymnesiophyceae and Pavlovophyceae. We detected pavlovophyte sequences only in freshwater systems as well as several novel prymnesiophyte phylotypes in both freshwater and marine environments. In addition, we retrieved a cluster of sequences (HAP-3) from the Marmara Sea branching deeply in the haptophyte tree with no clear affiliation to either of the two recognized classes. Five of the freshwater prymnesiophyte phylotypes detected formed a divergent monophyletic group (EV) without close described representatives that branched within the Isochrysidales, a group of generally marine and most often calcifying coccolithophorids. The presence of several sequences of freshwater haptophytes scattered among marine taxa in phylogenetic trees confirms the occurrence of several independent haptophyte transitions between marine and freshwater environments.