Abstract Study question What are the risk factors associated with mismatch (MM) and genuine mismatch (GMM) in the ART laboratory? Summary answer MM is associated with individual operators, oocyte denudation (OD) and embryo transfer (ET) processes, while GMM related to OD and extended work hours (>4pm). What is known already Mix-up of gametes or embryos in the ART laboratory causes dramatic consequences to both the patients and clinics. Recent studies using electronic witnessing system (EWS) equipped with radio frequency ID (RFID) tags, have revealed that most MM events only involved empty cell culture vessels, while the true mix-up risk arose from a subgroup of GMM involving culture vessels carrying gametes or embryos belonging to different patients. Exploration of risk factors associated with MM and GMM occurrence, separately, is still currently lacking in the literature. Study design, size, duration A total of 73719 witnessing points were retrospectively extracted from an EWS database, representing 11210 consecutive treatment cycles performed at Fertility North between January 2016 and July 2023. The 11210 treatment cycles comprised 5472 fresh IVF, 3732 frozen embryo transfer, 93 oocyte freeze, and 1913 intrauterine insemination cycles. Participants/materials, setting, methods All laboratory work areas were equipped with RFID readers. A MM was triggered by detection of RFID tagged cell culture vessels (either carrying biological material or not) and/or ID cards that belong to different patients in the same work area. A GMM was defined when a MM had involved biological materials and/or patients during procedure, with a potential mix-up. Risk factors evaluated included time of the day, weekdays, weekend, public holiday, operators, and laboratory processes. Main results and the role of chance A total of 138 (0.19%) MMs and 16 (0.02%) GMMs were identified during the study period, without actual mix-up. MM rate was the highest during 12-2pm (0.28%, p < 0.001) compared to other periods of the day, whilst GMM was the highest after 4pm (0.13%, p = 0.004). Neither MM nor GMM was associated with any specific weekday, weekend or public holiday (p > 0.05, respectively). Amongst 8 laboratory processes investigated, ET (0.36%, p < 0.001) led to the highest MM rate, followed by OD (0.35%, p = 0.021), but no increase was observed in oocyte collection, sperm preparation, insemination, embryo culture, cryopreservation and Assign ID. OD was the only process associated with higher GMM (0.13%, p < 0.001). Furthermore, Two of the 20 assessed operators were identified to produce more MMs (0.59%, p < 0.001, and 0.40%, p = 0.004; respectively) while GMM rates were similar among all operators (p > 0.05). Multivariate logistic regression indicated that the OD (adjusted odds ratio or aOR=2.147, p = 0.011) and ET (aOR=2.118, p < 0.001) processes as well as 2 individual operators (aOR=3.511, p < 0.001; aOR=2.538, p = 0.002; respectively) were independent risk factors to MM, but not the 12-2pm period of the day (aOR=1.341, p > 0.05). Finally, both extended work hours beyond 4pm (aOR=8.651, p = 0.005) and OD (aOR=9.538, p < 0.001) were independent risk factors to GMM. Limitations, reasons for caution Each individual ART laboratory has a unique team of scientists and workflow arrangement so findings in our study may not be universally applicable. GMM occurs at an extremely low frequency and is often calculated based on large numbers, therefore caution ought to be taken at statistical interpretation of data. Wider implications of the findings GMMs should be separated from MMs for analysis considering the very different clinical implications. Sufficient staffing with regular break and streamlined workflow are highlighted in preventing mix-up in the ART laboratory. Using electronically logged data, EWS enables identification of site-specific risk factors, which facilitates the development of corresponding preventative protocols. Trial registration number not applicable