There has recently been a great deal of interest in noninvasive high-frequency ultrasound imaging of small animals such as rats due to them being the preferred animal model for gene therapy and cancer research. Imaging rats using high-frequency ultrasound at frequencies above 30 MHz has become a crucial tool in many preclinical studies. Consequently, a clear understanding of the high frequency ultrasound backscattering and attenuation properties of rat blood is crucial for the design and future development of high frequency ultrasound Doppler and power Doppler imaging systems for rats. In the present study, the attenuation and backscattering coefficients of rat (Wistar rats) red blood cell (RBC) suspensions and whole blood with hematocrits ranging from 6% to 40% were measured between 30 and 60 MHz. Due to the focused transducers were used, a modified substitution approach was used for accurately measuring these ultrasonic parameters. The attenuation coefficients of rat blood were found to increase linearly with frequency and hematocrit. The average attenuation coefficient of rat whole blood with a hematocrit of 40% increased from 0.26 Nepers/mm at 30 MHz to 0.47 Nepers/mm at 60 MHz. The maximum backscattering coefficients of rat RBC suspensions were between 10% and 15% hematocrits at all frequencies. The fourth-power dependence of backscatter on frequency was approximately valid for rat RBC suspensions with hematocrits between 6% and 40%. The backscattering coefficient plateaued for hematocrits higher than 20% in rat whole blood, and shifted to a hematocrit of 10% at a higher speed. Due to the low RBC aggregation level in rat blood, the scattering properties of rat RBCs in plasma are similar to those of RBCs in saline at a higher rotation speed.