Background: miRNAs are known to play important regulatory roles throughout plant development. Until recently, nearly all the miRNAs in maize were identified by comparative analysis to miRNAs sequences of other plant species, such as rice and Arabidopsis.Results: To find new miRNA in this important crop, small RNAs from mixed tissues were sequenced, resulting in over 15 million unique sequences. Our sequencing effort validated 23 of the 28 known maize miRNA families, including 49 unique miRNAs. Using a newly established criterion, based on the precision of miRNA processing from precursors, we identified 66 novel miRNAs in maize. These miRNAs can be grouped into 58 families, 54 of which have not been identified in any other species. Five new miRNAs were validated by northern blot. Moreover, we found targets for 23 of the 66 new miRNAs. The targets of two of these newly identified miRNAs were confirmed by 5'RACE.Conclusion: We have implemented a novel method of identifying miRNA by measuring the precision of miRNA processing from precursors. Using this method, 66 novel miRNAs and 50 potential miRNAs have been identified in maize.