The latitudinal diversity gradient (LDG) is the most notable global biodiversity pattern, but its underlying mechanisms remain unresolved. The evolutionary speed hypothesis (ESH) posits that molecular rates play a crucial role in shaping the LDG, suggesting that higher temperatures accelerate molecular rates, thereby facilitating rapid speciation and accumulation of biodiversity in the tropics. However, whether ESH can explain the LDG across diverse taxonomic groups remains debated, and systematic examinations of its two key predictions using consistent datasets and methodologies across vertebrates are lacking. Here, we tested ESH using molecular rates from mitochondrial (5,424 species) and nuclear (1,512 species) genomes across major vertebrate groups, including fishes, amphibians, reptiles, mammals, and birds. Our findings revealed distinct latitudinal patterns in the absolute synonymous substitution rate (dS), which were influenced by thermoregulatory strategies. Specifically, the dS increases with ambient temperature and decreases with latitude in ectotherms but shows no correlation in most endotherms. These distinct patterns are likely attributed to different key predictors of dS between thermogroups, with temperature playing a major role only in ectotherms. For mitochondrial genes, absolute nonsynonymous substitution rates (dN) increase with temperature, likely driven by mutation rates in ectotherms and purifying selection in endotherms. However, neither mitochondrial dS nor dN correlates with diversification rates across vertebrates, contradicting the second prediction of ESH. For nuclear rates, the ESH was supported in reptiles and amphibians but not in mammals, birds, or fishes. In conclusion, our results provide limited support for ESH in vertebrates, underscoring the intricate processes that shape the LDG.