The objective of this study was to evaluate the effects of heat stress on the fecal microbiome of lactating dairy cows. We hypothesized there would be an increase in the richness and diversity of the bacterial communities in the feces of heat stressed cows. Six Holstein cows were housed in tie stalls in an environmental chamber. Cows averaged 175 ± 7 d in milk, 1.5 ± 0.5 parities and 36.3 ± 3.7 kg/d of milk and were fed and milked twice-daily. Cows were allowed 5-d acclimation to the chambers (d −5 to 0; temperature humidity index (THI)~65) and were then subjected to constant heat stress for 16d (d0 to 16; THI~76), followed by a 9d recovery period (d 16 to 24; THI~66). Feed and water were available ad libitum. Fecal samples were collected per rectum on d −1, 0, 6, 13, 16, 20 and 24. Samples were immediately frozen at −20°C. Fecal DNA was extracted using PowerFecal kits (Qiagen), the V4 hypervariable region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform, and operational taxonomic units (OTUs) were assigned to the SILVA database using BLAST based on a 97% nucleotide identity to evaluate richness and composition of fecal bacterial populations. Number of OTUs, and diversity assessed by the Shannon Index (Bonferroni t-test), increased approximately 20% and 5%, respectively, during heat stress compared with d −1 (P < 0.05, ANOVA). Diversity returned to d −1 levels by d 20 whereas the total number of OTUs was not fully restored until d 24. Principal component analysis (Bray-Curtis distances) revealed that community composition was similar during acclimation but diverged during heat stress and into the recovery period (P < 0.001, PERMANOVA). Results revealed individual OTUs displaying markedly different patterns of abundance across the experiment (P < 0.05). For example, compared with the acclimation period, Bacteroides species abundance increased 112% by d 16 of heat stress, then increased an additional 15% by d 24. We conclude that heat stress altered the fecal microbiome of lactating dairy cows affecting both diversity and abundance of individual OTUs. Altered fecal microbiome may impact gut health or environmental pathogen loads.