Hydrogen is an alternative source of energy of considerable interest, because it is environmentally friendly. Biological hydrogen production processes involving green microalgae are of significant interest. However, until present only few microalgae genera have been studied and almost all of those studies have focused only on cultivation using mixotrophic or heterotrophic media, which are expensive, and can be easily contaminated. This study aimed to compare the potential of biohydrogen production from novel green microalgae under autotrophic and mixotrophic media. A total of ninety strains of six orders of green microalgae were investigated for their capabilities of hydrogen production. The results showed that eleven novel hydrogen-producing microalgae genera were found. The hydrogen production in each order was influenced by the medium. Moreover, several strains presented notable levels of autotrophic hydrogen production and performed at over twice of the mixotrophic medium. These results should be supportive information for the selection and cultivation of hydrogen-producing microalgae in further studies.