In plants, directional transport of the hormone auxin creates concentration maxima and paths of transport that provide positional, polarity, and growth regulatory cues throughout development. In Arabidopsis thaliana, the polar-localized auxin transport protein PIN-FORMED1 (AtPIN1) is required to coordinate development during flowering. However, Arabidopsis has a derived PIN family structure; the majority of flowering plants have retained a clade of PIN proteins phylogenetically sister to PIN1, the Sister-of-PIN1 clade (SoPIN1), which has been lost in the Brassicaceae, including Arabidopsis. Based on PIN localization in the grasses Brachypodium distachyon and Zea mays, which have both SoPIN1 and PIN1 clades, we previously proposed that the organ initiation and vein patterning roles attributed to AtPIN1 were shared between the SoPIN1 and PIN1 clades in grasses. Here we show that sopin1 and pin1b mutants have distinct phenotypes in Brachypodium and thus the two clades are not functionally equivalent. sopin1 mutants have severe organ initiation defects similar to Arabidopsis atpin1 mutants, while pin1b mutants initiate organs normally but have increased stem elongation. Heterologous expression of Brachypodium PIN1b and SoPIN1 in Arabidopsis atpin1 mutants provides further evidence for functional distinction between the two clades. SoPIN1 but not PIN1b can complement null atpin1 mutants, while both PINs can complement an atpin1 missense allele with a single amino acid change. The different localization behaviors of SoPIN1 and PIN1b when heterologously expressed in Arabidopsis provide insight into how PIN accumulation at the plasma membrane, tissue-level protein accumulation, transport activity, and interaction, all contribute to the polarization dynamics that distinguish PIN family members. Combined, these results suggest that the PIN polarization and trafficking behaviors required for organ initiation differ from those required for other PIN functions in the shoot, and that in most flowering plants these functions are split between two PIN clades.