〔Abstract〕 To construct Csf1rCre Rosa26YFP mice, and to detect the efficiency of Csf1rCre -mediated yellow fluorescent protein (YFP) labeling monocytes and tissue-resident macrophages in different tissues. Methods The Rosa26YFP mutant mice had a loxP-flanked STOP sequence, followed by a yellow fluorescent protein gene (YFP) inserted into the Rosa26 locus. When crossed with mice expressing the Csf1rCre recombinase, the STOP sequence was deleted, and yellow fluorescent protein (YFP) expression was observed in the tissues of double-mutant offspring (Csf1rCre Rosa26YFP). Csf1rCre mice were mated with Rosa26YFP mice, and genotype of Csf1rCre Rosa26YFP mice were identified by PCR. Blood and bone marrow monocytes, liver macrophages, kidney macrophages, alveolar macrophages and spleen macrophages of adult Csf1rCre Rosa26YFP mice were isolated, labeled with flow cytometry antibodies, and the recombination efficiency of Csf1rCre-mediated YFP labeling tissue macrophages was analyzed by flow cytometry. Results In Csf1rCre Rosa26YFP reporter mice, the median percentage of YFP+ was 93.25% in renal macrophages, 92.45% in liver macrophages, and 91.10% in spleen macrophages. The percentage of YFP+ was 94.7% in alveolar macrophages, 98.2% in blood monocytes, and 93.9% (2.4%) in bone marrow monocytes. Conclusion Csf1rCre can be used to trace tissue-resident macrophages as well as bone marrow and blood monocytes. At the same time, Csf1rCre can target these cells to prepare conditional gene knockout mice.