Kisspeptin-GPR54 signalling is essential for normal reproductive functioning. However, the distribution of kisspeptin neuronal cell bodies and their projections is not well established. The present study aimed to provide a detailed account of kisspeptin neuroanatomy in the mouse brain. Using a polyclonal rabbit antibody AC566, directed towards the final ten C-terminal amino acids of murine kisspeptin, three populations of kisspeptin-expressing cell bodies were identified in the adult female mouse brain. One exists as a dense periventricular continuum of cells within the rostral part of the third ventricle, another is found within the arcuate nucleus, and another is identified as a low-density group of scattered cells within the dorsomedial nucleus and posterior hypothalamus. Kisspeptin-immunoreactive fibres were abundant within the ventral aspect of the lateral septum and within the hypothalamus running in periventricular and ventral retrochiasmatic pathways. Notable exclusions from the kisspeptin fibre innervation were the suprachiasmatic and ventromedial nuclei. Outside of the hypothalamus, a small number of kisspeptin fibres were identified in the bed nucleus of the stria terminalis, subfornical organ, medial amygdala, paraventricular thalamus, periaqueductal grey and locus coerulus. All kisspeptin cell body and fibre immunoreactivity was absent in brain tissue from Kiss1 knockout mice. These observations provide a map of kisspeptin neurones in the mouse brain and indicate that a limited number of mostly medial hypothalamic and lateral septal brain regions are innervated by the three hypothalamic kisspeptin cell populations; the functions of these projections remain to be established.