Nymphs of the hard tick Amblyomma americanum (L.) are an important life stage in the maintenance and transmission of tick-borne pathogens. As pathogen composition can vary across developmental cohorts, it is essential to understand the demographic structure of the questing population. Amblyomma americanum nymphs often display a second peak in activity during late summer, but it is unknown whether this peak represents older overwintered ticks or younger newly molted ticks. The objective of this study was to examine the heme concentration in field-captured A. americanum nymphs to determine if the questing population consists of one physiological cohort or multiple cohorts in a season. Ticks were collected from March to August in an old field of primarily non-native grasses, and heme concentration was used to assess physiological age. LOESS modeling depicted that heme concentration in the population declined from March to early July but increased in later sampling sessions. As ticks cannot replenish declining heme stores without a blood meal, a late-summer spike in heme concentration demonstrates that newly molted nymphs are entering the active population. The vector potential of these newly emerging nymphs may differ from those collected earlier in the year as pathogen diversity depends on reservoir host dynamics and timing of larval feeding.