ABSTRACT Hibiscus chlorotic ringspot virus (HCRSV) from naturally infected Hibiscus rosa-sinensis L. loses virulence in its experimental systemic host Hibiscus cannabinus L. (kenaf) after serial passages in a local lesion host Chenopodium quinoa . Here we report the genetic changes responsible for the loss of virulence at the molecular level. A remarkable covariation of eight site-specific amino acids was found in the HCRSV capsid protein (CP) after serial passages in C. quinoa : Val ⁴⁹ →Ile, Ile ⁹⁵ →Val, Lys ²⁷⁰ →Arg, Gly ²⁷² →Asp, Tyr ²⁷⁴ →His, Ala ³¹¹ →Asp, Asp ³³⁴ →Ala, and Ala ³³⁵ →Thr. Covariation of at least three of the eight amino acids, Val ⁴⁹ , Ile ⁹⁵ , and Lys ²⁷⁰ , caused the virus to become avirulent in kenaf. Interestingly, the nature of the covariation was consistent and reproducible at each serial passage. These data indicate that the nonsynonymous substitutions of amino acids in the HCRSV CP after serial passages in C. quinoa are not likely to be random events but may be due to host-associated positive selection or accelerated genetic drift. The observed interdependence among the three amino acids leading to avirulence in kenaf may have implications for structural or functional relationships in this virus-host interaction.