We present here an ultrastructure-based revision of the diatom genus Anorthoneis using both light (LM) and electron microscopy (SEM). The original slides and type material of all known Anorthoneis species were analysed and new, previously unseen, morphological features of the frustules, such as rota-like vela and ligulate structures around the valve margin, were revealed. The phylogenetic relationships among Anorthoneis taxa were investigated based on morphological characters using a maximum parsimony method. This analysis indicated a large, well-supported clade that included taxa that exhibited similarities in external and internal details of the central areas in both raphe-sternum and sternum valves. Revealed synapomorphies within the genus include externally and internally transversely dilated central area on both sides of the raphe-sternum valve, externally transversely dilated central area on both sides of the sternum valve, sternum valve striae parallel at the center and radiate at the apices, and areolae with hymenes in both valves. During a survey a new species Anorthoneis arthus-bertrandii sp. nov., was found on seagrass leaves collected from Siladen Island (Indonesia). This taxon is sister to A. hyalina, but can be easily differentiated from the latter by smaller valve dimensions, central area size and shape, and rota-like vela occluding sternum valve areolae - a feature that has never been observed in other Anorthoneis taxa.