MicroRNAs (miRNAs) negatively control gene expression by cleaving or inhibiting the translation of mRNA of target genes, and as such, they play an important role in plant development. Of the 79 plant miRNA families discovered to date, most are from the fully sequenced plant genomes of Arabidopsis, Populus and rice. Here, we identified miRNAs from leaves, roots, stems and flowers at different developmental stages of the basal eudicot species Eschscholzia californica (California poppy) using cloning and capillary sequencing, as well as ultrahigh-throughput pyrosequencing using the recently introduced 454 sequencing method. In total, we identified a minimum of 173 unique miRNA sequences belonging to 28 miRNA families and seven trans-acting small interfering RNAs (ta-siRNAs) conserved in eudicot and monocot species. miR529 and miR537, which have not yet been reported in eudicot species, were detected in California poppy; loci encoding these miRNAs were also found in Arabidopsis and Populus. miR535, which occurs in the moss Physcomitrella patens, was also detected in California poppy, but not in other angiosperms. Several potential miRNA targets were found in cDNA sequences of California poppy. Predicted target genes include transcription factors but also genes implicated in various metabolic processes and in stress defense. Comparative analysis of miRNAs from plants of phylogenetically-critical basal lineages aid the study of the evolutionary gains and losses of miRNAs in plants as well as their conservation, and lead to discoveries about the miRNAs of even well-studied model organisms.