To determine if progenies from the cross between Musa textilis Nee (2n = 20) and Musa balbisiana Colla (2n = 22) produced at the Institute of Plant Breeding (IPB), University of the Philippines Los Baños are true hybrids, abaca cultivars (Abuab and Inosa), a banana cultivar (Pacol), and their back crosses (BC₁, BC₂, and BC₃) were characterized morphologically and cytogenetically. Agronomic characters and fiber quality assessment through ANOVA and pairwise analysis revealed significant differences in tensile strength and agronomic parameters, namely, fiber quality and percent fiber recovery, except for leaf sheath number and girth measured at the top. This confirmed the recovery of recurrent parent genome (abaca) until the third generation of backcrosses. Moreover, this study reports the successful optimization of cytogenetic techniques. The most favorable time of root tips collection was from 10:00 a.m. to 10:30 a.m. due to the high number of dividing cells observed compared to other time slots tried. Two-hour cold shock pre-treatment resulted in considerably larger chromosomes, and higher number of well-spread prometaphase cells that helped in the construction of karyograms. Chromosome characteristics based on chromosome count and relative length were determined and compared among plant samples. Comparative karyotyping revealed a diploid chromosome number of 2n = 20 for abaca cultivars and hybrids. Inosa, another cultivar of M. textilis Nee, was observed to have a diploid chromosome number of either 2n = 20 or 2n = 22, in contrast to an earlier report of 2n = 17 to 2n = 23.