Aims: The objective was to determine the effects of dietary substitution of fishmeal (FM) with live yeast and increasing water temperature on the diversity and composition of gut microbiota in rainbow trout. Methods and Results: Fish were fed either FM or yeast (Saccharomyces cerevisiae) and reared in water temperatures of either 11 degrees C (cold) or 18 degrees C (warm) for 6 weeks. Luminal content and mucosa were collected from the distal gut and the load, diversity and species abundance of yeast and bacteria were analysed using agar plating, MALDI-TOF and rRNA gene amplicon sequencing. Yeast in the gut of fish fed FM were represented by S. cerevisiae, Rhodotorula spp. and Debaryomyces hansenii, while fish fed yeast contained 4-5 log higher CFU per g of yeast that were entirely represented by S. cerevisiae. For gut bacteria, sequencing of 16S rRNA gene amplicons using Illumina MiSeq showed lower bacterial diversity and abundance of lactic acid bacteria, especially Lactobacillus, in fish reared in warm rather than cold water. Fish fed yeast had similar bacterial diversity and lower abundance of Leuconostocaceae and Photobacterium compared with fish fed FM. Conclusions: Feeding live yeast mainly increased yeast load in the gut, while increased water temperature significantly altered the gut microbiota of rainbow trout in terms of bacterial diversity and abundance. Significance and Impact of the Study: Live yeast can replace 40% of FM without disrupting bacteria communities in the gut of rainbow trout, while increased water temperature due to seasonal fluctuations and/or climate change may result in a gut dysbiosis that may jeopardize the health of farmed fish.