The objective of this study was to re-analyse the molecular phylogeny and/or the morphology of all species, which have been attributed to the so-far mono-generic fungal family Ambisporaceae. The genus Ambispora has been well-known for its spore bi-morphy described even from single spore clusters. Triple-walled spores are differentiated on sporiferous saccules, while mono-walled spores are formed on simple subtending hyphae. New phylogenetic analyses revealed differences of ≥10% in nrDNA sequences of three phylogenetic clades, suggesting the need to divide Ambispora into three genera and perform advanced morphological separations. These advances are primarily based on the composition and phenotypic properties of the spore walls of the acaulo-ambisporoid morph, which are more diverse than those of the mono-walled glomoid morph. While all known species of the triple-walled acaulo-ambisporoid morph have an evanescent to semi-permanent outer spore wall, the middle wall of (i) Am. fennica, Am. brasiliensis, Am. gerdemannii and Am. nicolsonii is smooth and permanent (Am. fennica clade, A), (ii) Am. appendicula, Am. callosa, Am. leptoticha, and Am. jimgerdemannii is alveolate (Am. appendicula clade, B), and (iii) Am. granatensis is smooth and short-lived, easily degrading with age (Am. granatensis clade, C). Consequently, we decided that (i) species of the Am. fennica clade represent the genus Ambispora, (ii) species of the Am. appendicula clade represent the new genus Appendiculispora, and (iii) sequences previously ascribed to Am. granatensis represents a new clade at the rank of genus, here named Ephemeriparies. Two species of an additional morph, with triple-walled spores, but apparently formed on subtending hyphae, and having a diagnostic reticulate, football-like middle wall, are here separated from the revised genus Ambispora based solely on morphological analyses, since molecular identification analyses so far failed and remained merely unknown. This later morph and genus is based on the type species Pelotaspora reticulata comb. nov, and on P. austrolatina sp. nov. Concomitant molecular phylogenetic and morphological analyses are needed to attribute not only Pelotaspora species, but also those, for which hitherto only the glomoid-ambisporoid morph has been observed within the family Ambisporaceae. Without molecular analyses, such species with a glomoid morph recognized, but unknown acaulo-ambisporoid morph have to be retained within Ambispora.