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Abstract The pattern of gonadotropin secretion along the estrous cycle was elegantly described in rats. Less information exists about the pattern of gonadotropin secretion in gonad‐intact mice, particularly regarding the follicle‐stimulating hormone (FSH). Using serial blood collections from the tail‐tip of gonad‐intact C57BL/6 mice on the first day of cornification (transition from diestrus to estrus; hereafter called proestrus), we observed that the luteinizing hormone (LH) and FSH surge cannot be consistently detected since only one out of eight females (12%) showed increased LH levels. In contrast, a high percentage of mice (15 out of 21 animals; 71%) exhibited LH and FSH surges on the proestrus when a single serum sample was collected. Mice that exhibited LH and FSH surges on the proestrus showed c‐Fos expression in gonadotropin‐releasing hormone‐ (GnRH; 83.4% of co‐localization) and kisspeptin‐expressing neurons (42.3% of co‐localization) of the anteroventral periventricular nucleus (AVPV). Noteworthy, mice perfused on proestrus, but that failed to exhibit LH surge, showed a smaller, but significant expression of c‐Fos in GnRH (32.7%) and AVPVKisspeptin (14.0%) neurons. Finally, 96 serial blood samples were collected hourly in eight regular cycling C57BL/6 females to describe the pattern of LH and FSH secretion along the estrous cycle. Small elevations in LH and FSH levels were detected at the time expected for the LH surge. In summary, the present study improves our understanding of the pattern of gonadotropin secretion and the activation of central components of the hypothalamic–pituitary‐gonadal axis along the estrous cycle of C57BL/6 female mice.