The present study was designed to determine physic-chemical characteristics and best extender for refrigerated preservation of Labeo rohita milt. In experiment I, milt was collected from mature and healthy brooders (n = 5) of L. rohita reared at fish seed hatchery, Rawal Town, Islamabad, Pakistan. After collection, milt was maintained at 4°C and physico-chemical characteristics were studied. The mean volume of Labeo rohita milt was 8.02 ± 0.12 ml, pH 7.60 ± 0.17, sperm count 2.77 ± 0.19 1010/ml, osmolality 267.6 ± 7.4 mOsmol/kg, sodium 95.43 ± 6.7 (mM/L), potassium 34.41 ± 1.2 (mM/L), total lipids 178.94 ± 5.0 mg/dl and motility 83.5 ± 5.0%. In experiment II, nine male brooders (3 brooders/replicate) maintained in three different ponds were used. In each replicate, milt from three brooders was pooled, kept at 4°C and divided into 4 aliquots. An aliquot was kept undiluted (control), while other aliquots were diluted in Alsever's solution, sugar-based extender and modified Kurokura extender (KE) in 1:9 ratio at 4°C and stored up to 7 days. Sperm motility and viability were assessed for up to 7 days. Total antioxidant capacity was measured on Day 4. Sperm motility was higher (p < 0.05) in milt diluted with KE compared with control and other diluents from Day 5 to Day 7. However, highest sperm viability was observed on Day 6 and Day 7 in sperm diluted with KE. Total antioxidant capacity of milt diluted with KE and control was higher than other diluents on Day 4. In conclusion, viability of Labeo rohita sperm can be increased by diluting it in KE. [ABSTRACT FROM AUTHOR]