Neonatal maternal separation is a widely used method to construct an early-life stress model in rodents. In this method, pups are separated from their mothers for several hours every day during the first 2 weeks of life, which results in adverse early-life events. It is a known fact that maternal separation can exert a significant impact on the behavior and psychological health, such as anxiety and depression, in adolescent offspring. However, environmental conditions during maternal separation can differ such as the presence of other animals or by placing pups in a different dam. To investigate the differential effects of various conditions of maternal separation on the behavior of adolescent mice, we created the following groups: (1) iMS group: pups were moved to an isolated room with no other adult mice in a nearby cage, (2) eDam group: the pups randomly exchanged their dams, (3) OF group: pups were shifted to another cage with the bedding material containing maternal odor (olfactory stimulation), and (4) MS group: pups were shifted to another vivarium. From postnatal day (PND) 2–20 (i.e., 19 consecutive days), pups were separated from the dam daily for 4 h and exposed to various environments (MS, iMS, eDam, and OF) or were left undisturbed [control (CON) group]. A series of behavioral assessments were conducted to evaluate locomotion, anxiety, recognition, learning, and memory in adolescent offspring. The results showed that neonatal maternal separation led to impaired recognition memory, motor coordination, and motor skill learning across all groups. However, the iMS group exhibited anxiety-like behavior in the elevated plus maze test and enhanced the extinction of fear memory in the auditory fear conditioning test. The OF and eDam groups displayed partially recovered short-term working memory in the Y-maze test but exhibited opposite exploratory behaviors. The OF group spent more time in the center, while the eDam group spent less time. These findings demonstrated that exposure to different environmental conditions during maternal separation causes behavioral alterations in adolescent offspring, providing a potential explanation for the variation in behavioral phenotypes observed in the early-life stress models.