Frequently just prior to or at weaning (approximate age, 6 to 8 wk), puppies in research settings often develop diarrheal disease, which may be due, in part, to an immature and unstable intestinal microbiota that is permissive to opportunistic pathogens. The overall objective of this study was to assess whether fecal microbiota transfer (FMT) increased the transmission of a stable maternal microbiota to pups and decreased the incidence of postweaning diarrhea. Puppies were designated by litter as treated (FMT) or sham-treated. The FMT group received fecal inoculum orally for 5 consecutive days during weaning (at 6 to 8 wk of age). Diarrhea was evaluated according to a published scoring system for 11 d during the weaning period. Fresh feces were collected from dams and puppies at 3 d before weaning and 3, 10, and 24 d after weaning for analysis of the fecal microbiota by using 16S rRNA amplicon sequencing. The composition of fecal inoculum refrigerated at 3 to 5 °C was stable for at least 5 d. No diarrhea was reported in either group during the study period, making comparison of treated and control groups problematic. However, 16S rRNA gene analysis revealed microbial variability across time in both groups. Therefore, although the fecal microbiota of neither group of puppies mirrored the dam at any of the designated time points, the data provided fundamental and novel information regarding the dynamic maturation process of the fecal microbiota of puppies after weaning. Copyright 2016 by the American Association for Laboratory Animal Science.