Human sexuality is a complex reality, including gender identity and sexual orientation. A widespread approach to study human sexuality is to compare groups with opposite sexual approaches such as cisgenders vs transgenders and heterosexuals vs homosexuals. Neuroimaging studies have found brain differences between these groups of individuals. Nevertheless, they reported conflicting results and limitations such as small samples’ sizes and the considerable overlap between such groups makes it difficult to draw accurate conclusions. This systematic review explored structural, functional and metabolic features of the ‘cisgender brain’ compared with the ‘transgender brain’ before hormonal treatment and the ‘heterosexual brain’ compared to the ‘homosexual brain’ from the analysis of the neuroimaging literature up to 2018. Our main aim is to help identifying biological brain features that have been related to human sexuality to contribute to the understanding of the biological elements involved in gender identity and sexual orientation. Our results suggest that the majority of neuroanatomical, neurophysiological, and neurometabolic features in transgender individuals resemble those of their natal sex rather than those of their experienced gender and in homosexual individuals these resemble those of their same sex heterosexual population rather than their opposite sex heterosexual population. However, it is always difficult to interpret null findings with non-invasive neuroimaging. Given the gross nature of these measures, it is still possible that there are differences that are too subtle to measure with available tools yet have the impact of contributing to gender identity and sexual orientation. Moreover, conflicting results, also contributed to the impossibility of identifying specific brain features which consistently differ between cis- and transgender nor between hetero- and homosexual groups. The small number of studies, the small sample size of each study, and the heterogeneity of the investigations made impossible to meta-analyse all the data extracted. Further studies are necessary to increase the understanding of the neurological substrate of human sexuality.