The monarch butterfly (Danaus plexippus) is a vagile species that undertakes an annual, multi-generational migration across North America. The abundance of this species at both eastern and western overwintering sites in Central Mexico and California indicates a population decline. Success of continental-scale conservation programs for a migratory species depends on providing, maintaining, and protecting habitats at appropriate temporal and spatial scales. Here, dynamics of monarch continental-scale migration and gene flow were obtained by combined stable isotope, morphological, and genetic analyses. These analyses were applied to temporal monarch samples collected from May to September during 2016–2021 at locations in Iowa, USA and spatial collections from Pennsylvania, Delaware, Iowa, Ohio, Nevada, Idaho, Hawaii, 3 Australian locations during July and August 2016, andTexas in April 2021. Evidence of seasonal multi-generational migration was obtained through δ²H analyses of spatial collections, which was corroborated by decreased wing hue (a morphological marker for non-migratory individuals). In Iowa, 10–15% of monarchs represented migrants from southern areas throughout the breeding season and 6% were migrants from the North in midsummer. Limited sequence variation detected across the mitochondrial genome impacted the capability to detect significant population genetic variation in our North American samples. However, 2 novel substitutions were identified and predicted to be fixed among Australia samples, contributing to intercontinental differentiation from counterparts in North America. Our assessment of temporal and spatial population dynamics across the North American monarch breeding range provides insight into continental-scale migration and previously undetected mitochondrial DNA variation among globally distributed populations.