The stroke-prone spontaneously hypertensive rat (SHRSP) showed an exaggerated response to a high-fat high-cholesterol (HFC) diet, and the resulting reactive hypercholesterolemia was suggested to exacerbate the atherogenic process in this rat. We thus performed a QTL analysis on the serum cholesterol level of SHRSP before and after the HFC diet with the final goal being the identification of the genetic mechanisms of its reactive hypercholesterolemia. Three hundred and fifty-eight F2 rats between SHRSP and Wistar-Kyoto rat (WKY) were employed in the study. The serum cholesterol and apoprotein E were measured before and after 2-weeks of feeding with the HFC diet. Multiple QTLs for the basal cholesterol level were identified on chromosome 1 and 5, while those for the post-dietary cholesterol level were on chromosome 7, 15 and 16. The cholesterol QTLs before and after HFC diet did not overlap with one another, implying that the involved metabolic processes were considerably different between the two conditions. Supporting this, VLDL and LDL cholesterol were the major components of the post-dietary serum cholesterol, while the basal cholesterol level consisted mainly of HDL cholesterol. A substantial difference of the QTLs between males and females was observed, especially after the HFC diet. The QTL on chromosome 15 had an inverse effect on the cholesterol level, suggesting that the congenic substitution of the SHRSP fragment with that of WKY could induce a greater cholesterol level in SHRSP. This observation is significant in establishing a new model for atherosclerosis with hypertension in rats.