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Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms., Synopsis Transfer RNAs (tRNAs) translate the genetic code of genes into the amino acid sequence of proteins. Most amino acids have two or more codons. Every organism has multiple tRNA species reading the codons for the same amino acid (tRNA isoacceptors). In bacteria and yeast, differences in the relative abundance of tRNA isoacceptors have been found to affect the level of highly expressed proteins. This tRNA abundance–codon distribution relationship can have predictive power on the expression of genes based on their codon usages. Approximately 450 tRNA genes consisting of 49 isoacceptors and 274 different sequences have been annotated in the human genome. This work describes the first comparative analysis of tRNA expression levels in eight human tissues using microarray methods. The authors find significant, tissue-specific differences in the expression of tRNA species and coordinated expression among tRNAs decoding amino acids with similar chemical properties in distinct tissue types. Correlation of relative tRNA abundance versus the codon usage of highly expressed, tissue-specific genes can be found among a subset of tissues or tRNA isoacceptors. Differential tRNA expression in human tissues suggests that tRNA may play a unique role in regulating translation and possibly other processes in humans.