From a total genomic cosmid library of the pathogen Trypanosoma cruzi, specific sublibraries of the smallest four chromosomes were isolated by hybridization of the respective chromosomal bands obtained from pulsed-field gels. These libraries form the basis for initial mapping analyses that should provide information useful for both the ongoing physical mapping of the entire genome and eventual sequence analyses. Selectivity of the procedure was high with 75% to 92%, although cross-hybridization had to be expected from ubiquitous DNA features, such as centromeric and telomeric sequences, and other regions homologous between individual chromosomes. Overall, the number of identified clones was slightly higher than expected but well within the intrinsic experimental variation considering the uncertainty about the exact genome size, the variability in clonability and the higher frequency of repeat sequences in larger chromosomes. Chromosome III- and IV-specific cosmids were analyzed on Southern blots of chromosomal separations. For strain CL Brener, all clones tested exhibited cross-hybridization to a homologous chromosome larger than 1 Mbp, supporting the assumption of the respective chromosome couple being diploid pairs.