The order Prasiolales is one of the most characteristic lineages of the green algal class Trebouxiophyceae. Although its phylogeny has been studied in detail in recent years, several important features of its systematics and taxonomy are still in need of clarification. We investigated the phylogeny of the tufA gene in the Prasiolales, a marker that has been recently proposed for DNA barcoding in green algae but for which the amount of sequence data available is still limited for the Trebouxiophyceae. The results produced a phylogeny that was in good agreement with previous studies based on other markers, and the concatenation of tufA, rbcL and psaB resulted in a substantial improvement in phylogenetic resolution. Prasiola meridionalis and Prasiola stipitata, the two most common marine species of Prasiola in the northern hemisphere, had identical tufA sequences; we interpreted this genetic similarity to be the result of a trans-Arctic radiation related to the climatic history of the northern Atlantic and northern Pacific Oceans in recent evolutionary times. Sequences of the lichenized species Prasiola borealis indicate that the free-living form of this alga has a cosmopolitan distribution in cold temperate zones. In combination with other studies, our results suggested that the species of Prasiola commonly involved in associations with fungi were probably P. furfuracea and P. delicata rather than P. crispa, as was formerly believed. Based on the level of phylogenetic resolution of tufA in the Prasiolales, this gene may be a valuable phylogenetic marker at lower taxonomic levels in the Trebouxiophyceae and Ulvophyceae.