Abstract Early life stress leads to several effects on neurological development, affecting health and well-being later in life. Instances of child abuse and neglect are associated with higher rates of depression, risk taking behavior, and an increased risk of drug abuse later in life. This study used repeated neonatal separation of rat pups as a model of early life stress. Rat pups were either handled and weighed as controls or separated for 180min per day during postnatal days 2–8. In adulthood, male and female rats were tested for methamphetamine conditioned place preference reward and methamphetamine induced locomotor activity. Tissue samples were collected and mRNA was quantified for the norepinephrine transporter in the prefrontal cortex and the dopamine transporter in the nucleus accumbens. Results indicated rats given methamphetamine formed a conditioned place preference, but there was no effect of early separation or sex. Separated males showed heightened methamphetamine-induced locomotor activity, but there was no effect of early separation for females. Overall females were more active than males in response to both saline and methamphetamine. No differences in mRNA levels were observed across any conditions. These results suggest early neonatal separation affects methamphetamine-induced locomotor activity in a sex-dependent manner but has no effects on methamphetamine conditioned place preference.