The cultivation of horticultural plants in controlled greenhouse environments is a pivotal practice in modern agriculture, offering the potential to enhance crop productivity and mitigate climate change effects. This study investigates the biomechanical properties and lignin content of various Capsicum annuum mutant lines—‘fragile-plant’ (frx), ‘tortuous internodi’ (tti), and ‘puffy-structured stem’ (pfi)—in comparison to a commercially established variety, ‘Garai Fehér’. We employed the acetyl bromide method to quantify lignin content and conducted three-point bending tests to assess rigidity in three distinct regions of the stem. Gene expression analysis of key lignin biosynthetic pathway genes (PAL, C4H, 4CL, CCoAOMT, CAD) was performed using qRT-PCR. The results revealed significant differences in lignin content and breaking force among the genotypes and stem regions. The tti mutants exhibited similar lignin content to the control but lower breaking strength, likely due to elongated internodes. The frx mutants showed uniformly reduced lignin content, correlating with their fragile stems. The pfi mutants displayed abnormally high lignin content in the top region yet demonstrated the lowest stem rigidity in every region. Overexpression of CAD and CCoAOMT was detected in the mutants in specific regions of the stem, suggesting alterations in lignin biosynthesis; however, we could not confirm the correlation between them. Our findings indicate that while lignin content generally correlates with stem rigidity, this trait is complex and influenced by more factors.