Understanding genome and chromosome evolution is important for understanding genetic inheritance and evolution. Universal events comprising DNA replication, transcription, repair, mobile genetic element transposition, chromosome rearrangements, mitosis, and meiosis underlie inheritance and variation of living organisms. Although the genome of a species as a whole is important, chromosomes are the basic unit subjected to genetic events that coin evolution to a large extent. With complete genome sequences available, we can address evolution and variation of individual chromosomes across species. Here we show that chromosomes within a species do not show dramatic fluctuation in their content of mobile genetic elements as the proliferation of these elements increases from unicellular eukaryotes to vertebrates. Furthermore, we demonstrate that there is an upper limit to chromosome size variation in diploid eukaryotes with linear chromosomes. Strikingly, variation in chromosome size for 886 chromosomes in 68 eukaryotic genomes can be viably captured by a single model. This conserved boundary of chromosome size variation indicates that cellular, molecular, and evolutionary mechanisms, possibly together, confine the chromosome lengths around a species-specific average chromosome length. Analysis results with data from 14 newly sequenced species, containing a total of 179 chromosomes, further proved this finding.