Heterotrichous ciliates play an important role in aquatic ecosystem energy flow processes and many are model organisms for research in cytology,regenerative biology,and toxicology.In the present study,we combine both morphological and molecular data to infer phylogenetic relationships at family-genus level and propose new evolutionary hypotheses for the class Heterotrichea.The main results include: (1) 96 new ribosomal DNA sequences from 36 populations,representing eight families and 13 genera,including three poorly annotated genera,Folliculinopsis,Ampullofolliculina and Linostomella;(2) the earliest-branching families are Spirostomidae in single-gene trees and Peritromidae in the concatenated tree,but the family Peritromidae probably represents the basal lineage based on its possession of many “primitive” morphological characters;(3) some findings in molecular trees are not supported by morphological evidence,such as the family Blepharismidae is one of the most recent branches and the relationship between Fabreidae and Folliculinidae is very close;(4) the systematic positions of Condylostomatidae,Climacostomidae,and Gruberiidae remain uncertain based either on morphological or molecular data;and (5) the monophyly of each genus included in the present study is supported by the molecular phylogenetic trees,except for Blepharisma in the SSU rDNA tree and Folliculina in the ITS1-5.8S-ITS2 tree.