Mutation is the ultimate source of all genetic variation, and over the last ten years the ready availability of whole-genome sequencing has permitted direct estimation of mutation rate for many non-model species across the tree of life. In this meta-analysis we make a comprehensive search of the literature for mutation rate estimates in eukaryotes, identifying 140 mutation accumulation (MA) and parent-offspring (PO) sequencing studies covering 134 species. Based on these data, we revisit differences in single nucleotide mutation (SNM) rate between different phylogenetic lineages and update the known relationships between mutation rate and generation time, genome size, and nucleotide diversity—while accounting for phylogenetic non-independence. We do not find a significant difference between MA and PO in estimated mutation rates, but we confirm that mammal and plant lineages have higher mutation rates than arthropods, and that unicellular eukaryotes have the lowest mutation rates. We find that mutation rates are higher in species with longer generation times and larger genome sizes, even when accounting for phylogenetic relationships. Moreover, although nucleotide diversity is positively correlated with mutation rate, the gradient of the relationship is significantly less than one (on a logarithmic scale), consistent with higher mutation rates in populations with smaller effective size. For the 29 species for which data are available, we find that indel mutation rates are positively correlated with nucleotide mutation rates, and that short deletions are generally more common than short insertions. Nevertheless, despite recent progress, no estimates of either SNM or indel mutation rates are available for the majority of deeply-branching eukaryotic lineages—or even for most animal phyla. Even among charismatic megafauna, experimental mutation rate estimates remain unknown for amphibia and scarce for reptiles and fish. Lay Summary Over the past decade, the sequencing revolution has led to an ever-increasing number of mutation-rate estimates from mutation accumulation or parent-offspring sequencing studies in eukaryotes. However, studies rarely quantify to what extent the mutation rate varies among these species. Also, despite strong predictions as to how mutation rate should vary with (e.g.) generation time, there have been few recent or wide-ranging analyses of such predictors while accounting for the inherent similarity between closely-related species. Of particular note, there has been surprisingly little effort to robustly test the ‘drift barrier’ hypothesis that mutation rates should decrease with increasing effective population size. In this study, we used a comprehensive literature search to identify all the available experimental estimates of mutation rate in eukaryotes and subject them to phylogenetic mixed-model analyses. We find that per-nucleotide per-generation mutation rates differ by orders of magnitude among species: plants and mammals tend to have higher mutation rates than arthropods, and unicellular organisms have the lowest mutation rates. Our analysis also shows that mutation rates increase significantly with increasing generation time and genome size, and nucleotide diversity increases with mutation rate with a gradient less than one—as predicted by the drift-barrier hypothesis.