Previous studies have shown that exponentially growing Escherichia coli can detect mild acidity (~pH 5.5) and, in response, synthesize enzymes that protect against severe acid shock. This adaptation is controlled by the EvgS/EvgA phosphorelay, a signal transduction system present in virtually every E. coli isolate whose genome has been sequenced. Here we show that, despite this high level of conservation, the EvgS/EvgA system displays a surprising natural variation in pH-sensing capacity, with some strains entirely non-responsive to low pH stimulus. In most cases that we have tested, however, activation of the EvgA regulon still confers acid resistance. From analyzing selected E. coli isolates, we find that the natural variation results from polymorphisms in the sensor kinase EvgS. We further show that this variation affects the pH response of a second kinase, PhoQ, which senses pH differently from the closely related PhoQ in Salmonella enterica. The within-species diversification described here suggests EvgS likely responds to additional input signals that may be correlated with acid stress. In addition, this work highlights the fact that even for highly conserved sensor kinases, the activities identified from a subset of isolates may not necessarily generalize to other members of the same bacterial species.