Among-site rate variation, as quantified by the gamma-distribution shape parameter, a or alpha, and the ratio of transition rate to transversion rate (Ts/Tv) influence phylogenetic inference. We examine the effect of topology on estimates of these two parameters in 12S rRNA sequences from nine species of mice belonging to the genera Onychomys and Peromyscus by generating 100 random topologies and estimating these parameters using parsimony and maximum-likelihood methods for each of the random topologies. The parsimony-based estimate of Ts/Tv from the well-corroborated topology falls within the distribution of estimates based on random topologies, whereas the maximum-likelihood estimate of Ts/Tv based on the well-corroborated topology lies well outside the distribution of estimates derived from random topologies. The Ts/Tv ratio derived via maximum-likelihood estimation is three times the parsimony-based estimate, suggesting that parsimony-based estimates are severe underestimates even when the correct topology is used. Both parsimony- and likelihood-based estimates of the gamma-distribution shape parameter (alpha) are sensitive to topology because the best estimates based on the well-corroborated topology are well outside the distributions of estimates derived from random topologies for both methods. We show that the reason for topology dependence is the presence of long internal branches in the underlying topology.