Genome analysis is a global approach centered on a large-scale synthesis of genetic and physical mapping information and has emerged as one of the most effective tools for molecular genetics. Protozoan parasites have several common genomic features: (1) asexual or clonal growth is a normal part of the life cycle, (2) the haploid genome sizes are about the size of the smallest human chromosome, (3) the chromosomes can generally be resolved by pulsed-field gel electrophoresis (PFGE), (4) chromosome size polymorphisms among natural isolates are relatively frequent, and (5) the genomes appear to have relatively little dispersed-repetitive DNA. The trypanosomatids that include Trypanosoma and Leishmania species share some unusual properties: (1) all messenger RNAs are trans-spliced with a 5' spliced leader or miniexon 39-nucleotide sequence and (2) many genes are polycistronically transcribed and may exist as tandem arrays. Some of these characteristics can be used to tailor methods specifically for the analysis of protozoan parasite genomes. This chapter describes the way the ability to separate whole chromosomes by gel electrophoresis simplifies the assignation of genes to chromosomes, the way the large number of telomere ends may be used to generate chromosome tags, and the way the spliced leader sequence aids in the construction of a directionally cloned, full-length complementary DNA library. © 1995, Elsevier Inc.