Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are populations of non-T, non-B lymphocytes in peripheral tissues. Although NK and ILC1 subsets have been described, their identification and characteristics remain unclear. We performed single-cell RNA sequencing and CITE-seq to explore NK and ILC1 heterogeneity between tissues. We observed that although NK1 and NK2 subsets are conserved in spleen and liver, ILC1s are heterogeneous across tissues. We identified sets of genes expressed by related subsets or characterizing unique ILC1 populations in each organ. The syndecan-4 appeared as a marker discriminating murine ILC1 from NK cells across organs. Finally, we revealed that the expressions of EOMES, GZMA, IRF8, JAK1, NKG7, PLEK, PRF1, and ZEB2 define NK cells and that IL7R, LTB, and RGS1 differentiate ILC1s from NK cells in mice and humans. Our data constitute an important resource to improve our understanding of NK-ILC1 origin, phenotype, and biology. Keywords: CITE-seq; ILC1; NK; immunology; innate immunity; innate lymphoid cells; single-cell RNA-seq.