Resistant starch (RS) has been extensively studied because of its beneficial effects on gut microbiota. In this study, four RSs obtained through various preparation processes were utilized for in vitro fermentation, and their structural characteristics before and after fermentation were determined using chromatography, Fourier infrared spectroscopy, and scanning electron microscopy (SEM). It was observed that these RSs can be classified into two categories based on their fermentation and structural features. The autoclaving RS (ARS) and extruding RS (ERS) were classified as Class I Microbiome Community (MC-I), characterized by a higher proportion of butyrate and its producers, including unclassified_g_Megasphaera and Megasphaera elsdenii. While microwaving RS (MRS) and ultrasound RS (URS) belonged to Class II Microbiome Community (MC-II), marked by a higher proportion of acetate and its producer, Bifidobacterium pseudocatenulatum DSM 20438. MC-I had a lower molecular weight, shorter chain length, more chains with degree of polymerization (DP) 36-100, and a more ordered structure than MC-II. Furthermore, SEM observations revealed distinct degradation patterns between MC-I and MC-II, which may be attributed to their surface structural characteristics. These findings imply that the preparation methods employed for RS can determine its multilevel structural characteristics, and consequently influence its physiological properties.