A slower rate of starch digestion in the small intestine increases the amount of resistant starch (RS) entering the large intestine, which is associated with health benefits. Although increasing the amylose (AM) content of dietary starch intake is one way to increase RS, the processes involved in gut microbial hydrolysis and fermentation of high AM-RS substrates are poorly understood. In this study, five high AM wheat (HAW) starches ranging from 47% AM to 93% AM and a wild type (37% AM), in both native granular and cooked forms, were subjected to in vitro fermentation with a porcine faecal inoculum. Fermentation kinetics, temporal microbial changes, amylolytic enzyme activities and residual starch were determined. All granular starches showed similar fermentation characteristics, independent of AM level, whereas cooking accelerated fermentation of lower AM but slowed fermentation of high AM starches. HAW starches with a very high AM content (>85%) all had similar fermentation kinetics and short-chain fatty acid end-product profiles. Microbial α-amylase, β-amylase, pullulanase and amyloglucosidase enzymatic activities were all detected and followed fermentation kinetics. HAW starch promoted shifts in the microbial community, with increases of the family Lachnospiraceae and the genus Treponema observed, while the genera Prevotella and Streptococcus were reduced in comparison to 37% AM. Overall, these findings suggest that any HAW starch incorporated into high RS food products would be expected to have beneficial microbiota-mediated effects in terms of fermentation kinetics and end products.