Environmental isotopes can provide information about the composition of groups and the movement of people across landscapes. The archaeological record of Huron-Wendat communities in south-central Ontario is one of numerous drainage-based sequences of small villages among which families or larger population segments moved. These villages amalgamated in the early to mid-sixteenth century into fewer, larger communities. Strontium isotope values (87Sr/86Sr) are used to test hypotheses about these early localized interactions and later amalgamations. There is little prior information about strontium values from this region which was recently glaciated and receives ample precipitation. From the late thirteenth century onward, ancestral Huron-Wendat communities had distinct burial practices of primary burial followed by secondary, collective ossuary burial. Strontium values from tooth enamel of 118 human first permanent molars from 15 archaeological contexts spanning four centuries are interpreted in a framework of archaeologically derived deer (N = 34), small (N = 35) and other large (N = 7) mammals. Reflecting their local origins, 87Sr/86Sr values of small mammals from three geological substrates differ significantly from one another. Each small mammal group clusters more tightly than those of deer. Most human 87Sr/86Sr values agree with small mammal values, by region. Three sites, out of six with more than 10 data points, show mean human 87Sr/86Sr values that differ significantly from the small mammal values of their geological substrate, signaling community movement and individual in-migration. Interpretation of individual human values outside local ranges is informed by information from dietary isotopes. Environmental isotopes substantiate and enhance our prior understandings of the ancestral Huron-Wendat.