The cosmopolitan genus Ulva Linnaeus includes species of green macroalgae found in marine, brackish and some freshwater environments. Although there is a wide literature for the determination of Ulva taxa in Europe, they are among the most problematic algae to accurately identify, because they have few distinctive features, as well as a high intraspecific variation. At present, the knowledge of both diversity and distribution of the genus Ulva in the Mediterranean Sea is almost entirely based on morphological studies and there is only a few published papers dealing with molecular data. Tunisia has a key position in the Mediterranean and constitutes a transition area with a rich habitat diversity between eastern and western basins. The latest inventory of marine macrophytes dates back to 1987, updated in 1995. The aim of the present paper is to provide a molecular-assisted alpha taxonomy survey of Ulva spp. along Tunisian coasts, in comparison with a few Italian sites, using the tufA marker. Nine genetic species groups were resolved, including the non indigenous species Ulva ohnoi, newly reported for Tunisia. The actual picture of the taxonomy of Ulva spp. in the Mediterranean as a whole is far to be clarified and the present data on Tunisian collections aim to be a step towards its clarification. This paper is the first DNA barcoding study on green macroalgae in Tunisia and it contributes to add records to the Barcode Of Life Data Systems which are publically available.