Alternative sources of natural rubber are of interest due to economic, biological, and political threats to current supplies. Prickly lettuce (Lactuca serriola L.) is one of few species that synthesizes high molecular weight natural rubber. Genotypic analysis was conducted on an F2 segregating prickly lettuce population to discover genetic regions linked to natural rubber production. In total, 461 EST-SSRs were screened against the crossed parents. A genetic linkage map was created using 89 polymorphic markers. Three markers had no linkage and five dominant markers did not map leaving 81 mapped markers in 12 linkage groups. Seventeen phenotypic measurements were taken from four main categories, latex components, rubber qualities, leaf measurements, and growth patterns. Interval mapping (IM) and multiple QTL mapping (MQM) identified QTL that had LOD score significance. Rubber molecular weight had a QTL on linkage group 8 near markers WSULs-21, WSULs-374.2, and WSULs-210. Dispersity was linked to an unmapped marker, WSULs-374.1. Leaf perimeter and lobing had QTL on linkage group 8 near markers WSULs-102 and WSULs-212. Stem counts and growth habit had QTL on linkage group 4 near marker WSULs-304. Herbivory, plants that had been grazed or not grazed, had a surprising QTL on linkage group 8 between markers WSULs-12 and WSULs-21. The discovered QTL and corresponding markers are genetic resources for understanding rubber biosynthesis in prickly lettuce and useful in marker assisted selection (MAS) breeding. Prickly lettuce is an excellent candidate for elucidating the rubber synthesis mechanism and has potential as a crop plant for rubber production.