Peniculids comprise a large order of ciliated protists in Class Oligohymenophorea having many unresolved evolutionary relationships. Herein, we report 27 new sequences, including 18S rRNA, ITS1-5.8SITS2 rRNA, 28S rRNA and the mitochondrial cox1 genes of eight peniculids. We conducted phylogenetic analyses based on each these markers and on a four-gene concatenated data set (18S rRNA, ITS1-5.8SITS2 rRNA, 28S rRNA, and cox1 gene). The main findings are: 1) subclass Peniculia and family Parameciidae are monophyletic, with genus Frontonia remaining non-monophyletic; 2) Urocentrids have traditionally been regarded as a family, multi-gene analyses support the rank of Urocentrida and consistently recovers this order as sister to Peniculida, and Urocentrida and Peniculida comprise subclass Peniculia in agreement with Lynn's (2008) classification; 3) discrepancies between multiple-gene phylogenies, and conflicts with morphologic data regarding genus Frontonia necessitate expansion and revision of species diagnoses and we propose consideration of Group III of Frontonia (including F. didieri, F. ocularis, F. anatolica, F. pusilla and F. elegans) as incertae sedis in Peniculida; 4) multi-gene analyses of Parameciidae support five previously established subgenera. Paramecium buetschlii is placed in subgenus Chloroparamecium, and P. chlorelligerum into subgenus Viridoparamecium.