Geographic variation in acoustic signals can be important in species divergence, especially the maintenance of prezygotic barriers to gene flow. Furthermore, selective pressures on acoustic signals likely vary both across geographic distances and among vocalizations used in different behavioral contexts. We described the call repertoire of 5 subspecies of Marsh Wren (Cistothorus palustris) in eastern North America and tested for variation in both the acoustic structure and likelihood of production of each call type at 3 functional–ecological levels: subspecies identity, migratory pattern, and habitat type. Three of the 7 described call types exhibited acoustic variation best explained by either migratory pattern or habitat type. These calls were used principally in courtship–territorial patrol contexts, whereas 4 calls that did not exhibit geographic variation were used in agonistic interactions. How often a call is used in a population may be indicative of the behavior or breeding phenology associated with that vocalization. We found that 4 calls varied in how commonly they were produced among the subspecies and/or habitat types. We also described and quantified the degree to which these 5 subspecies produce calls in association with song—a little reported, but possibly more widespread, behavior in birds. Marsh Wrens commonly embedded 3 call types into song in a nonrandom pattern. This behavior was more common in freshwater-marsh populations than in saltmarsh populations, and we discuss several possible functions for call-song associations. Overall, when geographic variation in call structure occurred, it was most commonly explained by differences in habitat type and, therefore, may be indicative of local adaptations that could limit gene flow across environments.