Genomic In Situ Hybridization (GISH), a powerful tool to identify and to quantify genomic constituents in allopolyploids, has been widely used in plants but not in animals mainly due to technical problems in obtaining informative results. Using the allopolyploid Squalius alburnoides fish complex as a model system, we succeeded in overcoming methodological constraints when dealing with parental species with a small genome size. This hybridogenetic complex has biotypes with different genome compositions and ploidy levels, but parental chromosomes are small, morphologically very similar and therefore cannot be distinguished by conventional cytogenetic approaches. Specimens have a small genome (C-value = 1.2 pg) with a low level of highly and moderate repetitive sequences, mainly located at pericentromeric chromosome regions. Since it is well known that probe annealing depends on probe concentration and hybridization time to obtain uniform hybridization signals along the chromosome arms, we progressively increased the amount of labeled probes from 100ng up to 1µg per slide and the incubation time from overnight up to 72 h, among other minor improvements. Results showed a clear enhancement of signals with respect to previous data, allowing an accurate and reproducible assignment of the parental genomes in both diploid and triploid fish. It was thus evidenced that high probes’ concentrations and long incubation time are the key to obtain, without extra image editing, uniform and reliable hybridization signals in metaphase chromosomes of hybrid fish even involving parental species with small genome size.