The α‐actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α‐actinin‐1 and α‐actinin‐4 crosslink actin filaments in the cytoskeleton, while sarcomeric α‐actinin‐2 and α‐actinin‐3 serve a crucial role in anchoring actin filaments to the muscle Z‐line. To assess the difference in turnover dynamics and structure/function properties between the α‐actinin isoforms at the sarcomeric Z‐line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α‐actinin‐2/α‐actinin‐3 had the slowest turn over, α‐actinin‐1 recovered to an intermediate degree, and α‐actinin‐4 had the fastest recovery. Interestingly, the isoforms’ patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α‐actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z‐line. Protein domain‐specific investigations using α‐actinin‐2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N‐terminal actin‐binding domain, the spectrin‐like linker region and the C‐terminal calmodulin‐like EF hand domain. Together, these findings demonstrate that α‐actinin isoforms are unique in binding dynamics at the Z‐line and suggest differentially evolved interactive and Z‐line association capabilities of each functional domain.