Nowadays, cellulose nanocrystals (CNC) plays a major role in industrial processes due to their unique properties which, in turns, able to enhance the physical properties of bulk materials. The interstice properties of CNC are vary regarding to the source of the used cellulosic materials. The study aimed to characterize cellulose CNC extracted from two different genotypes of cotton slivers; Egyptian extra-long staple (G.88) and upland medium staple length (BF FK37). As per acid hydrolysis, CNC was extracted from different genotypes of cotton slivers by the use of H2SO4 6٠% (w/w) at 60 for 60 min. Then, the extracted CNC was characterized by making used of different tools e.g., transmission electron microscopy (TEM), Dynamic Light Scattering (DLS), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, Thermo Gravimetric analysis (TGA). Results revealed that, CNC was, successfully, extracted by acid hydrolysis of cotton slivers. Notably, the size of the obtained CNC was varied as genotypes of cotton slivers varies, which attained as shown from TEM; the average dimensions (diameter × length) of 36.46 ± 7 nm × 355 ± 98.3 nm for extra-long staple, 37.2 ± 14 nm × 284 ± 83 nm for medium staple. This fact was further supported by DLS as the size of extra-long staple was 22. 45 d.n and 35.77 d.n for medium staple. In addition, XRD results demonstrated that, both genotypes excited cellulose type I (crystalline polymorph). However, CNC extracted from extra-long showed higher crystallinity index (80%) than observed from medium staple. Moreover, the thermal stability observed for CNC of extra-long is significantly higher than obtained from medium. By varying of the source of CNC, the size of the obtained crystals was varied besides crystallinity index and their thermal stability properties. Therefore, the using of extra-long staple length cotton slivers led to reduce the crystal size with improving in both crystallinity and thermal properties