The neuropeptide kisspeptin, encoded by Kiss1, regulates reproduction by stimulating GnRH secretion. Neurons synthesizing kisspeptin are predominantly located in the hypothalamic anteroventral periventricular (AVPV) and arcuate nuclei, but smaller kisspeptin neuronal populations also reside in extrahypothalamic brain regions, such as the medial amygdala (MeA). In adult rodents, estradiol (E2) increases Kiss1 expression in the MeA, as in the AVPV. However, unlike AVPV and arcuate nuclei kisspeptin neurons, little else is currently known about the development, regulation, and function of MeA Kiss1 neurons. We first assessed the developmental onset of MeA Kiss1 expression in males and found that MeA Kiss1 expression is absent at juvenile ages but significantly increases during the late pubertal period, around postnatal day 35, coincident with increases in circulating sex steroids. We next tested whether developmental MeA Kiss1 expression could be induced early by E2 exposure prior to puberty. We found that juvenile mice given short-term E2 had greatly increased MeA Kiss1 expression at postnatal day 18. Although MeA Kiss1 neurons are known to be E2 up-regulated, the specific estrogen receptor (ER) pathway(s) mediating this stimulation are unknown. Using adult ERα knockout and ERβ knockout mice, we next determined that ERα, but not ERβ, is required for maximal E2-induced MeA Kiss1 expression in both sexes. These results delineate both the developmental time course of MeA Kiss1 expression and the specific ER signaling pathway required for E2-induced up-regulation of Kiss1 in this extrahypothalamic brain region. These findings will help drive future studies ascertaining the potential functions of this understudied kisspeptin population.