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To maintain and foster the welfare of laboratory mice, tools that reliably measure the current state of the animals are applied in clinical assessment. One of these is the Mouse Grimace Scale (MGS), a coding system for facial expression analysis. Since there are concerns about the objectivity of the MGS, we further investigated its reliability. Four observers (two experienced and two inexperienced in use of the MGS) scored 188 images of 33 female and 31 male C57BL/6JRj mice. Images were generated prior to, 150 min, and two days after ketamine/xylazine anesthesia. The intraclass correlations coefficient (ICC = 0.851) indicated good agreement on total MGS scores between all observers when all three time points were included in the analysis. However, interrater reliability was higher in the early post-anesthetic period (ICC = 0.799) than at baseline (ICC = 0.556) and on day 2 after anesthesia (ICC = 0.329). The best agreement was achieved for orbital tightening, and the poorest agreement for nose and cheek bulge, depending on the observers’ experience levels. In general, experienced observers produced scores of higher consistency when compared to inexperienced. Against this background, we critically discuss factors that potentially influence the reliability of MGS scoring.