The Hessian fly Mayetiola destructor (Diptera: Cecidmyiidae) is a major pest of wheat, globally. We conducted a series of laboratory choice and no-choice assays to quantify Hessian fly host preference for barley (cv. Champion), oat (cv. Cayuse), susceptible (cv. Alturas), and resistant (cv. Hollis) wheat. In addition, larval survivorship and adult emergence were compared among the evaluated host plants. We then examined whether insect preference for a host can be explained by differences in plant spectral reflectance. Further, larval survivorship and adult emergence were compared among host plants in relation to phytohormone concentrations. Hessian flies laid more eggs on wheat compared to either oat or barley. Spectral reflectance measurements of leaves were similar between susceptible and resistant wheat cultivars but different from those of barley and oat. Our results suggested that higher reflectance in the near-infrared range and lower reflectance in the visible range may be used by females for host selection. Hessian fly larvae were unable to develop into the pupal stage on resistant wheat and oat. No significant difference in larval survivorship was detected between the susceptible wheat and barley. However, adult emergence was significantly higher on barley than the susceptible wheat. Phytohormonal evaluations revealed that salicylic acid (SA) may be an important contributor to plant defense response to larval feeding as relatively higher concentrations of SA were present in oat and resistant wheat. While resistance in the resistant wheat is achieved only through antibiosis, both antibiosis and antixenosis were in effect rendering oat as a non-host for Hessian flies.