Abstract: Microbiota from mothers is an essential source of microbes in early-life rumen microbiota, but the contribution of microbiota from different maternal sites to the rumen microbiota establishment in neonates needs more data. To fill this gap, we collected samples from the mouth, teat skin, and rumen of lactating yaks and from the rumen of sucking calves concomitantly on seven occasions between days 7 and 180 after birth under grazing conditions. We observed that the eukaryotic communities clustered based on sample sites, except for the protozoal community in the teat skin, with negative correlations between fungal and protozoal diversities in the rumen of calves. Furthermore, fungi in the dam’s mouth, which is the greatest source of the calf’s rumen fungi, accounted for only 0.1%, and the contribution of the dam’s rumen to the calf’s rumen fungi decreased with age and even disappeared after day 60. In contrast, the average contribution of the dam’s rumen protozoa to the calf’s rumen protozoa was 3.7%, and the contributions from the dam’s teat skin (from 0.7 to 2.7%) and mouth (from 0.4 to 3.3%) increased with age. Thus, the divergence in dam-to-calf transmissibility between fungi and protozoa indicates that the foundation of these eukaryotic communities is shaped by different rules. This study provides the first measurements of the maternal contribution to the fungal and protozoal establishment in the rumen of sucking and grazing yak calves in early life, which could be beneficial for future microbiota manipulation in neonatal ruminants. Key points: • Dam to calf transfer of rumen eukaryotes occurs from multiple body sites. • A minor proportion of rumen fungi in calves originated from maternal sites. • The inter-generation transmission between rumen fungi and protozoa differs.