Bamboo, a member of subfamily Bambusoideae in the grass family (Poaceae), is one of the most important nontimber forest resources and a potential alternative to wood and wood products. Ethiopian lowland bamboo (
Oxytenanthera abyssinica
) is an economically and ecologically important species which accounts about 85% of total bamboo coverage in the country. This species is experiencing population decline due to a number of anthropogenic factors. As a foundation step, genetic diversity, population structure, and gene flow analysis of various
O. abyssinica
populations found in Ethiopia are studied using inter‐simple sequence repeat markers. One hundred and thirty isolates of bamboo belonging to 13 geographically diverse populations were collected for DNA extraction and analysis. Heterozygosity, level of polymorphism, marker efficiency, Nei's gene diversity (
H
), and Shannon's information index (
I
) analysis, analysis of molecular variance (AMOVA), analysis for cluster, principal coordinates (PCoA), and admixture analyses were performed based on the markers banding pattern. The results indicated high genetic variation (84.48%) at species level. The
H
,
I
, observed and effective number of alleles at the species level were 0.2702, 0.4061, 1.8448, and 1.4744, respectively, suggesting a relatively high level of genetic diversity. However, genetic differentiation at the population level was relatively low. Using grouped populations, AMOVA revealed that most (61.05%) of the diversity was distributed within the populations with
F
ST
= 0.38949,
F
SC
= 0.10486, and
F
CT
= 0.31797. Cluster analysis grouped the populations into markedly distinct clusters, suggesting confined propagation in distinct geographic regions. STRUCTURE analyses showed
K
= 2 for all populations and
K
= 11 excluding Gambella population. Using these markers, we found strong evidence that the genetic diversity of the lowland bamboo is associated with distinct geographic regions and that isolates of Gambella Region, with their unique genetic origin, are quite different from other bamboos found in the country.