The K+ channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K+ binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K+-selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K+ channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K+ conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K+ channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K+ channels. Our results establish the generality of K+-induced SF conformational stability across the K+ channel superfamily, and introduce an approach to study manipulation of channel selectivity.