Despite high volumes of manure production in the Shenandoah Valley, there are still areas of forage production in the Shenandoah Valley that are nutrient deficient, because manures have traditionally been applied to higher value row crops. Furthermore, anecdotal reports have suggested that application of poultry litter to pastureland may increase the abundance of weeds. This study was conducted to compare the effects of poultry litter and inorganic fertilizers on soil fertility, aboveground botanical composition and soil seed bank composition of established mixed naturalized pastures. Two sites each had the following treatments applied for two consecutive years: 1) split application of litter: 3.36 Mg ha-1 in spring and 3.36 Mg ha-1 in summer; 2) inorganic fertilizer at same N, P and K rates as Trt. No. 1; 3) single application of litter: 6.72 Mg ha-1 in spring; 4) inorganic fertilizer at same N, P and K rates as Trt. No. 3; 5) unfertilized control. Forage yield, soil pH, Mehlich 1 P (M1P), P balance, water soluble phosphorus (WSP), shifts in aboveground botanical composition, seed bank composition, and species richness (SPR) were monitored during this experiment. In addition, we examined whether poultry litter carries germinable weed seeds that can be transferred to forage systems after application. On average, soil pH was 0.2 higher for litter than chemical fertilizer treatments, but this difference was not significant. Mehlich 1 P and WSP both increased throughout the two years, but no significant differences were found amongst split and single application or litter and inorganic fertilizers. Phosphorus balance analysis indicated that forage yield did not remove adequate P to prevent environmental concerns, when using N based applications. Fertilization increased yield 3.5 Mg ha-1 on average and yield responses to application timing and fertilizer types were similar. Desirable species abundance increased significantly (linear regression, P< 0.05) over time in both fertilizer and poultry litter application treatments. Furthermore at one site, Shenandoah County, poultry litter application actually reduced the number of weedy species in plots (P=0.04). Weedy plant abundance did not differ at either site, and no significant change in species richness (number of species/area) was observed at Rockbridge County for any treatment. No treatment effects were found for seed bank species richness and number of germinated seeds (m-2). No seedlings germinated directly from poultry litter collected from several sites in Virginia. Poultry litter was comparable to inorganic fertilizer in terms of increasing soil WSP, M1P, forage yield, and above ground composition of desirable forage species. Timing of application made no difference. Additionally, our results indicate that poultry litter has no viable seed and does not increase weed abundance in mixed naturalized pastures.