Post-translational modifications (PTMs), such as phosphate groups, control many cellular activities and play important roles in both health and disease. Accordingly, there is an urgent need for affinity reagents that target modified sites on individual proteins and can be used in applications ranging from immunodetection to immunotherapy. However, the generation of synthetic PTM-binders remains a major challenge using traditional hybridoma or selection methods, and the resulting affinity reagents are often nonspecific. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The genetic assay was validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). Using the selection strategy, we performed affinity maturation of a phospho-specific DARPin and uncovered several superior binders including a protein with 70-fold improved affinity (to 0.15 nM) for its cognate antigen, pERK2, but with no significant change in affinity for non-cognate ERK2. The selection strategy was similarly applied to reprogram the specificity of the same DARPin, yielding promiscuous variants that evolved the ability to bind non-cognate ERK2 but that still retained binding to pERK2. Collectively, these results establish our PTM-specific genetic selection as a useful and potentially generalizable new tool for studying PTM-specific binding proteins and customizing their affinity and selectivity.