Plant nuclear genomes encompass a wide range of variation in size and nucleotide composition with diverse arrangements of chromosomal segments, repetitive sequences and distribution of genes. Comparative genomic analysis may be undertaken at different levels of organisation, which are reflected in this review, together with a focus on the genetic and functional significance of the observed variation. Patterns of genome organisation have been revealed which reflect the different underlying mechanisms and constraints driving change. Thus comparative issues of genome size, nucleotide sequence composition and genome heterogeneity are provided as a background to understanding the different levels of segmental and repetitive sequence duplication and distribution of genes. The extent of synteny and collinearity revealed by recent genetic and sequence comparisons is discussed, together with a consideration of problems associated with such analyses. The possible origins and mechanisms of variation in genome size and organisation are covered, including the prevalence of duplication at different levels of organisation. The likely genetic, functional and adaptive consequences of replicated loci are discussed with evidence from comparative studies. The scope for comparative analysis of epigenetic plant genome variation is considered. Finally, opportunities for applying comparative genomics to isolating genes and understanding complex crop genomes are addressed.