<p>Despite numerous studies on the tensile properties of banana fibres, no comprehensive investigation has yet been reported into the factors responsible for the significant variations in tensile strength (TS) across different banana varieties. To address this research gap, the TS of fibres from eight varieties representing five diverse genomic groups (BB, AAA, ABB, ABB, AAAA) was measured, and various test methods were applied to critically study the variations. Physico-chemical analyses included component analysis, linear density, Fourier-transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy for both fibre surface and cross-sectional morphologies. Additionally, X-ray diffraction was conducted to investigate the cellulose-related factors, including cellulose crystallinity, crystallite size and d-spacing. The TS varied significantly from 61 to 367 MPa; however, the relative proportions of ‘A’ and ‘B’ genomes, cellulose content, crystallite size and cellulose crystallinity did not directly support this trend. In contrast, the d-spacing values strongly negatively corroborated with TS (r = − 0.75), revealing that the variations in the orderly arrangement of crystalline cellulose I allomorphs are crucial in determining TS of banana fibres. Moreover, the fibre microstructure, particularly cell wall thickness and well-defined middle lamella, also positively influenced TS, but only when allomorphs with denser structural packing arrangements predominated. Hence, this suggests a complex interaction between different cellulose I allomorphs and fibre microstructural features, which is further influenced by the predominance of genome ‘A’ in the banana A/B hybrids. The identification of key factors determining TS of banana fibres, marks a significant advancement in addressing this knowledge gap.</p>