Tossa jute (Corchorus olitorius L.) is a natural fibre crop that produces good quality golden fibre. Predicting fibre yield and quality is crucial in jute breeding programs. Fibre anatomical characteristics provide valuable insights into both fibre yield and quality. Hence, seven tossa jute genotypes were investigated through field-based morphological studies and laboratory-oriented anatomical studies for fibre yield and yield-attributing morpho anatomical variability at Bangladesh Jute Research Institute (BJRI) during March 2023 to February 2024. Morphological analysis revealed significant genotypic variations in plant height, base diameter, green weight, fibre yield and stick yield (p < 0.05), highlighting their potential as key differentiators for breeding activities. The genotypes JRO-524 and O-0412-9-4 exhibited superior traits, including higher plant height (2.67 m and 2.64 m) and base diameter (13.89 mm and 13.18 mm) at 110 days after sowing. High heritability was observed for green weight (80.22 %) and fibre yield (80.05 %), indicating that these traits can be effectively targeted for genetic improvement. Anatomical studies revealed significant variability in stem diameter, bark thickness and fibre bundle attributes (p < 0.01). JRO-524 demonstrated the highest stem diameter (17.67 mm) and bark thickness (1.53 mm), while BJRI Tossa pat 9 excelled in trapezoid width and total bundle area. Principal component analysis (PCA) captured 85.7 % of cumulative variance across the first three components, with plant height, base diameter and fibre yield being major contributors. The PCA biplot categorized genotypes into five groups, elucidating genetic diversity for targeted breeding strategies. These findings will provide a foundation for developing high-yielding, fibre-rich tossa jute varieties in future.