The Salix alba L. (white willow)—Salix fragilis L. (crack willow) complex includes closely related polyploid species, mainly tetraploid (2n = 4x = 76), which are dioecious and hence obligate allogamous. Because little is known about the genome constitution and chromosome behavior of these pure willow trees, genetic analysis of their naturally occurring interspecific polyploid hybrids is still very difficult. A two-way pseudo-testcross strategy was exploited using single-dose AFLP markers in order to assess the main inheritance patterns of tetraploid biotypes (disomy vs. tetrasomy) in segregating populations stemmed from S. alba × S. fragilis crosses and reciprocals. In addition, a genomic in situ hybridization (GISH) technology was implemented in willow to shed some light on the genome structure of S. alba and S. fragilis species, and their hybrids (allopolyploidy vs. autopolyploidy). The frequency of S. alba-specific molecular markers was almost double compared to that of S. fragilis-specific ones, suggesting the phylogenetic hypothesis of S. fragilis as derivative species from S. alba-like progenitors. Cytogenetic observations at pro-metaphase revealed about half of the chromosome complements being less contracted than the remaining ones, supporting an allopolyploid origin of both S. alba and S. fragilis. Both genetic segregation and genomic hybridization data are consistent with an allotetraploid nature of the Salix species. In particular, the vast majority of the AFLP markers were inherited according to disomic patterns in S. alba × S. fragilis populations and reciprocals. Moreover, in all S. alba against S. fragilis hybridizations and reciprocals, GISH signals were observed only on the contracted chromosomes whereas the non-contracted chromosomes were never hybridized. In conclusion, half of the chromosomes of the pure species S. alba and S. fragilis are closely related and they could share a common diploid ancestor, while the rest of chromosomes are morphologically differentiated in either S. alba or S. fragilis and they should derive from distinct diploid ancestors.