In recent decades, Lyme disease has been expanding to previous nonendemic areas. We hypothesized that infected I. scapularis nymphs that retain host-seeking behavior under optimal environmental conditions are fit to fulfil their transmission role in the enzootic cycle of B. burgdorferi. We produced nymphal ticks in the laboratory under controlled temperature (22-25°C), humidity (80-90%), and natural daylight cycle conditions to allow them to retain host-seeking/questing behavior for 1 year. We then analyzed differences in B. burgdorferi infection prevalence in questing and diapause nymphs at 6 weeks postmolting (prime questing) as well as differences in infection prevalence of questing nymphs maintained under prolonged environmental induced questing over 12 months (prolonged questing). Lastly, we analyzed the fitness of nymphal ticks subjected to prolonged questing in transmission of B. burgdorferi to naive mice over the course of the year. B. burgdorferi infected unfed I. scapularis nymphal ticks maintained under optimal environmental conditions in the laboratory not only survived for a year in a developmental state of prolonged questing (host-seeking), but they retained an infection prevalence sufficient to effectively fulfil transmission of B. burgdorferi to uninfected mice after tick challenge. Our study is important for understanding and modeling Lyme disease expansion into former nonendemic regions due to climate change. IMPORTANCE Lyme disease is rapidly spreading from its usual endemic areas in the Northeast, Midwest, and Midatlantic states into neighboring areas, which could be due to changing climate patterns. Our study shows that unfed I. scapularis nymphal ticks kept under optimal environmental conditions in the laboratory survived for a year while exhibiting aggressive host-seeking behavior, and they maintained a B. burgdorferi infection prevalence which was sufficient to infect naive reservoir hosts after tick challenge. Our study raises important questions regarding prolonged survival of B. burgdorferi infected host-seeking nymphal I. scapularis ticks that can potentially increase the risk of Lyme disease incidence, if conditions of temperature and humidity become amenable to the enzootic cycle of B. burgdorferi in regions currently classified as nonendemic.