Low-order streams contribute to the abiotic and biotic character of large rivers and are renowned for harboring unique forms of aquatic flora and fauna. However, most studies on headwater streams mainly focus on the mainstems and overlook the contribution of the tributary systems. Moreover, low-order streams are generally overlooked in legislation and bioassessment programs, and consequently not protected in many countries. To contribute to the recognition of the ecological importance of low-orders streams, this study focused on determining whether river network characteristics and associated physico-chemical parameters can be used to effectively predict the variabilities in macroinvertebrate assemblage characteristics between first-order and second-order streams in the headwaters of the Nzoia River Basin, Kenya. The study quantified the structural and functional community composition, diversity, similarity, and richness of macroinvertebrate communities between the two river systems. Dissolved oxygen, coarse particulate organic matter, conductivity, stream width, depth, discharge and flow velocity were the main predictors of the diversity and distribution of macroinvertebrates in the first order and second-order streams. The first order streams recorded higher abundance of macroinvertebrates than the second-order streams. Taxa from families Ephydridae, Elmidae, Gomphidae, and genera Euthraulus, Neoperla, Orthothrichia and Prosopistoma were limited to the second-order stream sites while families Ceratopogonidae, Pisuliidae, Dytiscidae and genus Trichosetodes occurred exclusively in the first-order stream sites. Collector-filterers and collector-gatherers were the most abundant functional feeding groups (FFGs) in the two river systems. Scrapers were abundant in the second-order stream while shredders were abundant in the first order streams. The distinctness in the structural and functional composition of macroinvertebrates between the two river systems suggests that linkages among streams in a network as exemplified in the Nzoia River Basin, support and foster biodiversity.