Symbiotic associations are of a broad significance in evolution and biodiversity. The process of endosymbiosis is one of promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells harbors the individuals of unicellular photoautotrophic algae. We reconstructed the phylogeny of algal endosymbionts isolated from green hydra strains collected from four different geographical sites within Croatia and one from Germany and one from Israel. Nuclear (18S rRNA, the ITS region), chloroplast (16S rRNA, rbcL) and mitochondrial (COI) markers for maximum likelihood phylogenetic analyses were used. We focused on investigating the positions of these algal endosymbiotic strains within the chlorophyte lineage. Molecular analyses established that different genera and species of unicellular green algae from class Trebouxiophyceae and Chlorophyceae are present as endosymbionts in green hydra, showing that are of polyphyletic origin. Our results indicate that the intracellular algal endosymbionts of green Hydra have become established several times independently in evolution. Isolated endosymbiotic algae were characterized by light microscopy and scanning and transmission electron microscopy, according to cytological morphometric parameters and by activity and isoenzyme analysis of catalase, peroxidase and esterases. The results obtained by isoenzyme analysis suggest a difference between Croatian and European algal endosymbionts, indicating biological diversity among algal symbionts.