The genus Lambertella is currently considered to be one of the largest genera in the family Rutstroemiaceae, and its major distinguishing character is the pigmentation of ascospores, besides a substratal stroma. Although Lambertella appears to be well-defined by morphological characters, its phylogenetic heterogeneity has been suggested in earlier studies. To circumscribe the genus more precisely, morphological examination and phylogenetic analysis was conducted on some Lambertella species and some allied stroma-forming fungi. In total, 58 taxa were included in the study, including 16 species with both a substratal stroma and pigmented ascospores. The polyphyly of Lambertella sensu lato was confirmed based on the phylogenetic analysis of ITS, LSU, and RPB2 regions. A highly supported clade is composed of five Lambertella species, including the type L. corni-maris. Morphologically, all species here included in Lambertella s. s. have brown ascospores prior to being discharged from asci, whereas in other species currently placed in Lambertella, but now excluded from the genus, they turn brown only after discharge. This clade was defined as Lambertella sensu stricto. Pigmentation of the ascospores was also recognized in Lanzia and Poculum, and even in the helotiaceous genus Hymenoscyphus, but always only after discharge. The convergent evolution of a dark substratal stroma in Rutstroemiaceae and Helotiaceae was also indicated.