Multiply inverted balancer chromosomes that suppress exchange with their 
homologs are an essential part of the Drosophila melanogaster genetic toolkit. 
Despite their widespread use, the organization of balancer chromosomes has not 
been characterized at the molecular level, and the degree of sequence variation 
among copies of balancer chromosomes is unknown. To map inversion breakpoints 
and study potential diversity in descendants of a structurally identical 
balancer chromosome, we sequenced a panel of laboratory stocks containing the 
most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the 
locations of FM7 breakpoints to precise euchromatic coordinates and identified 
the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP 
variation revealed megabase-scale blocks of sequence divergence among currently 
used FM7 stocks. We present evidence that this divergence arose through rare 
double-crossover events that replaced a female-sterile allele of the singed gene 
(sn(X2)) on FM7c with a sequence from balanced chromosomes. We propose that 
although double-crossover events are rare in individual crosses, many FM7c 
chromosomes in the Bloomington Drosophila Stock Center have lost sn(X2) by this 
mechanism on a historical timescale. Finally, we characterize the original 
allele of the Bar gene (B(1)) that is carried on FM7, and validate the 
hypothesis that the origin and subsequent reversion of the B(1) duplication are 
mediated by unequal exchange. Our results reject a simple nonrecombining, clonal 
mode for the laboratory evolution of balancer chromosomes and have implications 
for how balancer chromosomes should be used in the design and interpretation of 
genetic experiments in Drosophila.