BackgroundTransporting pregnant cattle that have passed 90% or more of the expected gestation period (G90 threshold) is prohibited within the European Union. Therefore, there is a need to determine whether this threshold has been exceeded in late-gestation cows sent to slaughter. The aim of this study was to evaluate fetal parameters’ reliability for use in forensic age assessment of late-term Holstein fetuses.ResultsAnalysis of the gestation length of 2734 Holsteins that calved with a single liveborn fetus revealed a median gestation length of 278 days with 99% of parturitions occurring between day 261 and 290, corresponding to G90 thresholds of 235 and 261 days, respectively. The association between gestation length and neonatal body weight had an R2of 0.27. The influence of fetal sex and cow parity on gestation length was ± 2 days. The eruption of incisor and canine teeth was assessed in preterm calves delivered by caesarean section (n = 52) and full-term neonatal calves (n = 54). Statistical analysis of tooth eruption data showed a statistically significant variation in fetal age at tooth eruption.ConclusionsDefining the G90 threshold for a cow not having reached parturition is challenging. Body weight was not found to be a reliable parameter for identifying fetuses beyond the G90 threshold. Statistical analysis of the association between fetal age and eruption through the gingival mucosa of incisor and canine teeth revealed significant variation, making tooth eruption a challenging parameter to use in forensic cases. Assessment of the evaluated parameters, therefore, cannot be considered a scientifically validated method to conclude definitively and beyond reasonable doubt whether or not a given fetus has passed the G90 threshold.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13028-023-00691-0.