The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage. Here, we compare three transgenic mouse strains in which GFP is considered "ubiquitously expressed," with the GFP transgene under control of the chicken ␤-actin (CBA) or human ubiquitin C (UBC) promoter. We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available anti-GFP antibodies. Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood. Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney. UBC-GFP mice express GFP only weakly in solid organs and variably in blood. Direct fluorescent detection of GFP in formalin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence. Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence. Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine. In conclusion, there is considerable variability of expression within and between GFP transgenic strains. None of the tested strains gave truly ubiquitous GFP expression. A detailed analysis of GFP expression in one's tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin.