Aim: Whole genome sequencing and functional annotation of Bacillus nealsonii strain AAU1, an amylolytic anaerobic spore forming isolate from ruminal contents of buffalo. Methods and Results: Morphologically, the strain was observed as slender Grampositive rods, occurring in pairs. Optimal growth was observed at 40°C (range: 30°C to 45°C) and pH 6.5 (range: 5.5 to 7.5) when cultivated in Hungate’s medium supplemented with starch. The microorganism showed extracellular constitutive amylolytic activity, proving to be capable of utilizing glucose, maltose, mannose, trehalose, dextrin and starch under anaerobic conditions. Sequence analysis revealed a GC content of 35.1 mol%. Comparison of housekeeping gene sequences for RNA polymerase subunit B (rpoB) and gyrase A (gyrA) identified sequence similarity within the Bacillus genus, confirmed by 16S rRNA gene sequence similarity which identified Bacillus nealsonii DSM 15077 as the closest publically available relative. Chemotaxonomic analysis provided conflicting results with straight-chain saturated C16: 0/C16:0 aldehyde, C16:0 DMA, C14:0 and monounsaturated 16:1w7c and 16:1w9c the major fatty acids in contrast to those reported for B. nealsonii DSM15077. Further characterization using AN-Bilog and physiological parameters provided genotypic and phenotypic support for taxonomic classification of isolate AAU1 with published Bacillus species including B. licheniformis, B. subtilis, B. circulans and B. nealsonii. Conclusion: Based on the data presented, isolate is likely to represent a new strain/subspecies, for which the identifier B. nealsonii AAU1 is proposed. Significance and Impact of Study: The strict anaerobic conditions prevailing in the bovine rumen from where AAU1 was isolated may have resulted in genetic polymorphism influencing its metabolic characteristics.