The taxonomic rank and phylogenetic relationships of the pipizine flower flies (Diptera: Syrphidae: Pipizini) were estimated based on DNA sequence data from three gene regions (COI, 28S and 18S) and 111 adult morphological characters. Pipizini has been treated as a member of the subfamily Eristalinae based on diagnostic adult morphological characteristics, while the larval feeding mode and morphology is shared with members of the subfamily Syrphinae. We analysed each dataset, both separately and combined, in a total evidence approach under maximum parsimony and maximum likelihood. To evaluate the influence of different alignment strategies of rDNA 28S and 18S genes on the resulting topologies, we compared the topologies inferred from a multiple alignment using fast Fourier transform (MAFFT) program with those topologies resulting from aligning the secondary structure of these rDNA genes. Total evidence analyses resolved pipizines as a sister group of the subfamily Syrphinae. Although the structural alignment and the MAFFT alignment differed in the inferred relationships of some clades and taxa, there was congruence in the placement of pipizines. The homogeneous morphology of the Pipizini clade in combination with their unique combination of characters among the Syrphidae suggest a change of rank to subfamily. Thus, we propose to divide Syrphidae into four subfamilies, including the subfamily Pipizinae stat. rev.