To date, the influence of lignin variation on starch accumulation, composition and processing properties in storage roots remains underexplored. We used physiological and biochemical assay to estimate the influence of lignin on starch formation and pasting profiles in 21 cassava storage root species, along with a lignin detection assay using Raman spectroscopy. The lignin content ranged from 3.9 to 7.5% across the species, and the monolignol types primarily consisted of syringyl (S) and guaiacyl (G) units with S/G ratios of 0.16–0.96. The starch content varied greatly among the varieties (26.5–86.9%), with the amylose contents of 12.6–23.9%. Lignin content was negatively correlated with starch content and its composition (P < 0.01) in cassava. The rapid viscosity analysis curves revealed large variations in cassava flour and starch among the varieties. Starch pasting properties gradually decreased with increasing lignin content. The peak height of the Raman spectrum at 1603 cm−1 was positively correlated with lignin content (R2 = 0.96, P < 0.01), which can be used for the detection of lignin content in cassava. Overall, lignin content exhibited a negative impact on starch formation, composition, and pasting profiles. Breeding of lignin-free varieties is a prospective strategy for specific food and bioenergy production from cassava.