Background: A mismatch between organizational priorities and system-level policies can negatively impact implementation and sustainment of classroom-based physical activity (PA) interventions. The purpose of this study was twofold: (1) present methods to systematically identify organization- and system-level implementation barriers, and (2) align organizational priorities and system policies by designing multi-level implementation strategies. This alignment will support implementation scale-up of a tailored PA intervention in one low-resource intermediate school district (ISD; 16 districts, 32 schools) in central Michigan. Methods: Multi-level assessments of organizational readiness were conducted using the Hexagon Discussion and Analysis Tool to assess intervention-context fit, the Wellness School Assessment Tool 3.0 to evaluate district PA policy strength and comprehensiveness, and semi-structured interviews were conducted to assess administrative support and priorities related to PA programming. Results: Our assessments revealed three implementation barriers: limited structural capacity to sustain teacher training, limited resources across districts and school buildings to support teachers, and misalignment of ISD and district PA policies and priorities. Conclusions: Greater attention to organizational capacity and existing infrastructure should be considered "a priori" to support effective implementation and sustainment of PA interventions in low-resource schools.