The microbiome and associated metabolome of faecal samples were compared to those from the caecum and right dorsal colon of horses and ponies euthanised for nonresearch purposes by investigating the microbial population community structure as well as their functional metabolic products. Through the use of 16S rRNA gene dendrograms, the caecum microbiome was shown to cluster separately from the other gut regions. 16S rRNA gene-based quantitative PCR (q-PCR) also demonstrated differences between the caecum and the other gut regions. Metabolites as identified by Fourier transform infrared clustered in a similar way and specific metabolic products (volatile fatty acids and ammonia) also varied by region. Protozoal 18S rDNA concentration and archaeal mcrA gene concentration quantified by q-PCR were found in higher numbers in the colon than the other gut regions. Diversity calculations using Simpson and Shannon-Wiener indices demonstrated higher diversity in the right dorsal colon and faeces than in the caecum. All findings of this study suggest that faecal samples are likely to represent the microbial population of the right dorsal colon to some extent but not that of the caecum, indicating careful consideration is required when planning microbial investigations of the hindgut of the horse.