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Many non-model rodent species are inaccessible to genetic engineering due to our limited understanding of their reproductive biology. Here, we present a low-cost, camera-based estrous-tracking technology that enables transgenesis in the white-footed mouse Peromyscus leucopus, a key reservoir for Lyme disease. We demonstrate the efficient generation of pregnant and pseudopregnant mice via timed ovulation, provide protocols for embryo generation, cultivation, microinjection, and transplantation as well as an accurate developmental timeline, and report the first engineered Peromyscus. The same technology successfully tracked conserved estrous-linked cycling behavior in other rodents, including hamsters. Finally, estrous tracking differentiated reproductively healthy, geriatric female Peromyscus from those with declining fertility based solely on their activity, providing a non-invasive method for studying reproductive senescence. Collectively, these tools represent a critical resource for engineering non-model rodents, advance the long-lived Peromyscus as a model organism, and will prove essential to heritably immunizing wild rodent populations against Lyme disease.