We report a novel three-dimensional (3D) ultrashort echo time (UTE) sequence employing Cones trajectory and T1ρ preparation (UTE-Cones-T1ρ ) for quantitative T1ρ assessment of short T2 tissues in the musculoskeletal system. A basic 3D UTE-Cones sequence was combined with a spin-locking preparation pulse for T1ρ contrast. A relatively short TR was used to decrease the scan time, which required T1 measurement and compensation using 3D UTE-Cones data acquisitions with variable TRs. Another strategy to reduce the total scan time was to acquire multiple Cones spokes (Nsp ) after each T1ρ preparation and fat saturation. Four spin-locking times (TSL = 0-20 ms) were acquired over 12 min, plus another 7 min for T1 measurement. The 3D UTE-Cones-T1ρ sequence was compared with a two-dimensional (2D) spiral-T1ρ sequence for the imaging of a spherical CuSO4 phantom and ex vivo meniscus and tendon specimens, as well as the knee and ankle joints of healthy volunteers, using a clinical 3-T scanner. The CuSO4 phantom showed a T1ρ value of 76.5 ± 1.6 ms with the 2D spiral-T1ρ sequence, as well as 85.7 ± 3.6 and 89.2 ± 1.4 ms for the 3D UTE-Cones-T1ρ sequences with Nsp of 1 and 5, respectively. The 3D UTE-Cones-T1ρ sequence provided shorter T1ρ values for the bovine meniscus sample relative to the 2D spiral-T1ρ sequence (10-12 ms versus 16 ms, respectively). The cadaveric human Achilles tendon sample could only be imaged with the 3D UTE-Cones-T1ρ sequence (T1ρ = 4.0 ± 0.9 ms), with the 2D spiral-T1ρ sequence demonstrating near-zero signal intensity. Human studies yielded T1ρ values of 36.1 ± 2.9, 18.3 ± 3.9 and 3.1 ± 0.4 ms for articular cartilage, meniscus and the Achilles tendon, respectively. The 3D UTE-Cones-T1ρ sequence allows volumetric T1ρ measurement of short T2 tissues in vivo.