Latex is widely found among plant species and is known to play a defensive role against certain herbivores. Two romaine lettuce, Lactuca sativa L. (Asteraceae) cultivars, ‘Valmaine’ (resistant) and ‘Tall Guzmaine’ (susceptible), were selected to study the potential of latex as a defense mechanism against the banded cucumber beetle, Diabrotica balteata LeConte (Coleoptera: Chrysomelidae). Latex from Valmaine strongly inhibited feeding of adult D. balteata compared to latex from Tall Guzmaine when applied to the surface of artificial diet. Beetles consumed significantly less diet from disks treated with Valmaine latex than they consumed from diet treated with Tall Guzmaine latex, in both choice and no‐choice tests. In a choice test involving diet disks treated with Valmaine latex from young leaves vs. disks treated with latex from mature leaves, the beetles consumed significantly less diet treated with latex from young leaves. No significant difference in diet consumption was found between diets treated with latex from young and mature leaves of Tall Guzmaine in choice tests. Three solvents of differing polarity (water, methanol, and methylene chloride) were tested to extract deterrent compounds from latex; Valmaine latex extracted with water:methanol (20:80) strongly inhibited beetle feeding when applied to the surface of artificial diet. In no‐choice tests, fewer beetles were observed feeding on diet treated with water:methanol (20:80) extract of Valmaine latex than on diet treated with a similar extract of Tall Guzmaine latex, resulting in significantly less consumption of the diet treated with the Valmaine latex extract. These studies suggest that moderately polar chemicals within latex may account for resistance in Valmaine to D. balteata.