As of April 2018, new legislation in the EU requires food manufacturers to put in place steps to mitigate acrylamide formation in their products. Acrylamide is a known carcinogen that forms naturally during baking. One possible solution for reducing acrylamide formation in bread is the reduction of the main precursor of acrylamide, the amino acid asparagine. In wheat, this requires the use of select agronomic practices, along with the development of new varieties with lower propensity to form free asparagine. Hence, levels of free asparagine in Canadian wheat need to be determined, and mitigated if necessary, to maintain access to key export markets including Europe. To address this concern, a comprehensive study of the effect of genotype, growing location and fertilizer treatments on the free asparagine content of eight registered and commercially grown Canadian wheat varieties was undertaken. Wholemeal flours from CWRS (6), CPSR (1) and CNHR (1) varieties grown at two locations in Manitoba (Carberry and Lilyfield) under four fertilizer treatments were assessed for free asparagine content. For this purpose, an ultrahigh pressure liquid chromatograph (UPLC) equipped with a photodiode array detector was used. Statistical analysis of data was conducted using Generalized Linear Model for analysis of variance (ANOVA). A significant effect (P<0.05) of genotype (G) on asparagine levels in wheat was observed, indicating the need for effective breeding strategies to develop varieties with intrinsically lower levels of asparagine. A significant effect of location was also observed, with significantly higher free asparagine levels in wheat samples grown in Carberry compared to those grown in Lilyfield. Furthermore, the effect of genotype×es; location was found to be significant, with ranking of varieties for asparagine content differing between the two growing locations. Further investigations on the relationships between fertilizer treatments, free asparagine levels in Canadian wheat varieties, dough strength and acrylamide levels in bread are underway. Such studies will benefit Canadian wheat breeders, producers, exporters, and policy makers by providing sound quantitative data on important traits of Canadian wheat.