Summary Lettuce produces natural rubber (NR) with an average Mw of > 1 million Da in laticifers, similar to NR from rubber trees. As lettuce is an annual, self‐pollinating, and easily transformable plant, it is an excellent model for molecular genetic studies of NR biosynthesis. CRISPR/Cas9 mutagenesis was optimized using lettuce hairy roots, and NR‐deficient lettuce was generated via bi‐allelic mutations in cis‐prenyltransferase (CPT). This is the first null mutant of NR deficiency in plants. In the CPT mutant, orthologous CPT counterparts from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were expressed under a laticifer‐specific promoter to examine how the average Mw of NR is affected. No developmental defects were observed in the NR‐deficient mutants. The lettuce mutants expressing guayule and goldenrod CPT produced 1.8 and 14.5 times longer NR, respectively, than the plants of their origin. This suggests that, although goldenrod cannot synthesize a sufficiently lengthy NR, goldenrod CPT has the catalytic competence to produce high‐quality NR in the cellular context of lettuce laticifers. Thus, CPT alone does not determine the length of NR. Other factors, such as substrate concentration, additional proteins, and/or the nature of protein complexes including CPT‐binding proteins, influence CPT activity in determining NR length.