A conodont fauna of late Tremadocian to early Floian age (Early Ordovician) is documented from the Yinchufu Formation of Zhejiang Province, South China. It is characterized by the occurrence of two species of Prioniodontidae, a new species of Prioniodus and Acodus triangularis. Prioniodus antiquus sp. nov. may represent the most primitive species of Prioniodus, which is one of the earliest conodont genera with a ramiform—pectiniform apparatus. Based on the review of nearly 200 species originally assigned to Prioniodus, six multielement species are confirmed to belong to this genus. Morphological changes of these six species shows that they form an evolutionary lineage directly evolved from an adentate species, likely Acodus triangularis which has been reported from South China, Australia and from the Precordillera of western Argentina. Origination of the ramiform—pectiniform apparatuses as represented by the appearances of Prioniodus through late Tremadocian to Floian of the Early Ordovician might be a major response of the 'conodont animals' to occupy and adapt to the increasingly diversified environments in the shelf and slope settings. The biofacies distribution of the six Prioniodus species indicates that Prioniodus might have originated in deep-water slope settings and progressively spread into distal and then interior shelves in the late Tremadocian and diversified in the Floian, with Prioniodus amadeus possibly representing a relict species that survived into the Middle Ordovician and was restricted to shallow water environments.