Acrylamide, a suspected human carcinogen, is generated during food processing at high temperatures in the Maillard reaction, which involves reducing sugars and free asparagine. In wheat derivatives, free asparagine represents a key factor in acrylamide formation. Free asparagine levels in the grain of different wheat genotypes has been investigated in recent studies, but little is known about elite varieties that are cultivated in Italy. Here, we analysed the accumulation of free asparagine in a total of 54 bread wheat cultivars that are relevant for the Italian market. Six field trials in three Italian locations over two years were considered. Wholemeal flours obtained from harvested seeds were analysed using an enzymatic method. Free asparagine content ranged from 0.99 to 2.82 mmol/kg dry matter in the first year, and from 0.55 to 2.84 mmol/kg dry matter in the second year. Considering the 18 genotypes that were present in all the field trials, we evaluated possible environment and genetic influences for this trait. Some cultivars seemed to be highly affected by environment, whereas others showed a relative stability in free asparagine content across years and locations. Finally, we identified two varieties showing the highest free asparagine levels in our analysis, representing potential useful materials for genotype x environment interaction studies. Two other varieties, which were characterized by low amounts of free asparagine in the considered samples, may be useful for the food industry and for future breeding programs aimed to reduce acrylamide-forming potential in bread wheat.