The complete genome of a maize chlorotic mottle virus isolate from Nebraska (MCMV-NE) was cloned and sequenced. The MCMV-NE genome consists of 4,436 nucleotides and shares 99.5 nucleotide sequence identity with an MCMV isolate from Kansas (MCMV-KS). Of 22 polymorphic sites, most resulted from transition with a clear bias for U to C and C to U substitutions. The MCMV-NE genome was assembled into a single plasmid insert and used as a template to transcribe RNA in vitro. As RNA transcribed from the cloned MCMV-NE genome was infectious to maize plants, sequence differences between MCMV-NE and MCMV-KS are most likely neutral with respect to pathogenicity and virulence.