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High and variable pre-weaning mortality is a persistent problem in laboratory mouse breeding. Assuming a modest 15% mortality rate across mouse strains, means that approximately 1 million more pups are produced yearly in the EU to compensate for those which die. This paper presents the first large study under practical husbandry conditions to determine the risk factors associated with mouse pre-weaning mortality. We analysed historical records from 219,975 pups from two breeding facilities, collected as part of their management routine and including information on number of pups born and weaned per litter, parents’ age and identification, and dates of birth and death of all animals. Pups were counted once in their first week of life and at weaning, and once every one or two weeks, depending on the need for cage cleaning. Dead pups were recorded as soon as these were found during the daily cage screening (without opening the cage). It was hypothesized that litter overlap (i.e. the presence of older siblings in the cage when new pups are born), a recurrent social configuration in trio-housed mice, is associated with increased newborn mortality, along with advanced dam age, large litter size, and a high number and age of older siblings in the cage. The estimated probability of pup death was two to seven percentage points higher in cages with litter overlap compared to those without. Litter overlap was associated with an increase in death of the entire litter of five and six percentage points, which represent an increase of 19% and 103% compared to non-overlapped litters in the two breeding facilities, respectively. Increased number and age of older siblings, advanced dam age, small litter size (less than four pups born) and large litter size (over 11 pups born) were associated with increased probability of pup death. This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/ 2020 and by FEDER -Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 –Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT -Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/CVT-WEL/ 1202/2014 (POCI-01-0145-FEDER-016591)